Novel antibody-drug conjugates and the use of same in therapy

ABSTRACT

Disclosed are novel antibody-drug conjugates and use thereof in therapy, in particular in anticancer or anti-inflammatory therapy, as well as synthetic products useful as linkers, composed of a linker head and a linker body, and also a method for preparing the linkers and the antibody-drug conjugates.

The present invention relates to novel antibody-drug conjugates and use thereof in therapy, in particular in anticancer or anti-inflammatory therapy. The present invention also relates to synthetic products useful as linkers, composed of a linker head and a linker body. The present invention also relates to a method for preparing the linkers and the antibody-drug conjugates.

An antibody-drug conjugate is a new form of drug, in particular used as an anticancer agent with targeted action. The therapeutic antibody binds to one or more cell types as a function of its specificity and conventionally releases a cytotoxic agent in the cells.

An antibody-drug conjugate constitutes a means for selective delivery of a cytotoxic agent. This engineering therefore makes it possible to combine the specificity of targeting by antibodies with powerful new effector functions by the agents with which they are conjugated.

To date, more than 20 antibody-drug conjugates are under development (Senter, P. D. et al. Annu. Rev. Med. 2013, 64, 15-29).

The general structure of an antibody-drug conjugate is as described in FIG. 1.

The product linking the antibody and the drug is called a linking agent or linker. It can be grafted onto the antibody via at least one of the eight cysteines forming the 4 interchain disulphide bridges or onto at least one of the eight lysines.

The number of drug molecules grafted onto the antibody determines a ratio called the Drug-Antibody Ratio (DAR).

After binding to its target antigen, the antibody is internalized in the cell by receptor-mediated endocytosis. The vesicles fuse with lysosomes where the cytotoxic molecule is released from the antibody by various mechanisms. The active cytotoxic agent then acts directly on the cell, inducing its death, and sometimes on the neighbouring cancer cells by transport or diffusion in the environment.

The antibody is therefore used mainly as a vector and delivers the cytotoxic agent into the cell.

By “cytotoxic agent” is meant a molecule capable of inhibiting or preventing the function of a cell.

The cytotoxic agent bound to the antibody is a prodrug that becomes active in the tumour cell after release (Jaracz et al. 2005). The activity of the free molecule must be sufficient to kill the cancer cell, even at low concentration.

Despite their growing success, antibody-drug conjugates have a notable drawback, because they are more complex and have a heterogeneous structure compared to the corresponding original antibody. Attachment of a cytotoxic agent via a linker has a significant influence on the pharmacokinetics and pharmacodynamics of the antibody (PK-PD) and therefore on the therapeutic index.

The antibody-drug conjugates preferably have an average DAR equal to 4 for optimum activity but this ratio can vary from 1 to 13, knowing that for a DAR there can be a population of distinct entities.

Many researchers are working on this heterogeneity. Tests have been carried out with the aim of modifying the sequence of the antibody, by introducing natural or non-natural amino acids (Mallet, W. et al. Nat. Biotechnol. 2008, 26, 925-932 and Schultz, P. G. et al. Proc. Natl. Acad. Sci. 2012, 109, 16101-16106). Nevertheless, this method has a disadvantage, that of having to be transposed for each antibody of interest, which requires consistent work on the starting antibody.

Consequently, the technical problem that arises from the prior art is to obtain a technological means allowing controlled grafting of a cytotoxic agent without modifying the sequence of the antibody of interest—a technological means that can be adapted to any antibody.

One aspect of the invention is a synthetic product useful as a linker head.

Another aspect of the invention is a product defined as a linker head, bound to an element L representing a linker body.

Another aspect of the invention is a linker bound to an element M defined as a cytotoxic drug.

Another aspect of the invention is a linker associated with a cytotoxic drug and associated with a protein, which can in particular be an antibody or an antibody fragment.

The present invention is mainly based on a product that is characterized in that it corresponds to formula I selected from formulae IB and IA:

in which: X is a halogen, or a nucleofuge, or a

group, or a

group, X_(a) being a halogen or a nucleofuge.

A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic,

or A represents a

group, a

group or a

group X₁ is a C═O or a single bond X₂ is an NH group or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0, 1, 2 or 3; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, W represents

-   -   an —OH radical,     -   an -L radical which represents a linker body comprising a         terminal reactive function,     -   an -FL fluorophore group used in diagnostics or analysis,         involving detection of fluorescence preferably selected from         rhodamine or a derivative thereof, preferably rhodamine B,         fluorescein isothiocyanate (FITC), a Cy dye preferably selected         from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected         from Alexa Fluor 647, 700 or 750, Texas Red, allophycocyanin         (APC) and conjugates thereof with other fluorochromes in         particular those mentioned above, phycoerythrin (PE) and         conjugates thereof with other fluorochromes in particular those         mentioned above,     -   an R* radioactive radical, which preferably contains         either a bifunctional ligand, optionally bimodal, preferably         selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA,         NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA;         or a bifunctional ligand preferably selected from one of the         acyclic chelating agents from the list constituted by EDTA,         CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA,         BOPA;         or a bifunctional ligand preferably selected from one of the         macrocyclic chelating agents from the list constituted by DOTA,         TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP,         DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide,         p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA,         NOTA, CHX-A″-DTPA;         or a bifunctional chelating agent of the cationic, anionic,         neutral or cleavable type;     -   or a radionuclide preferably selected from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y,         ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,         ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁷Au,         ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm, ⁴⁴Sc, ⁴⁷Sc,         ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb, ²¹²Bi, ²¹³Bi,         ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd, ¹⁴⁸Nd, ⁸⁹Sr,         ²¹¹At,     -   an -L-M radical in which L has the meaning given above and M         represents a cytotoxic drug selected from a chemotherapeutic         agent or a toxin,         P is a protein comprising at least one disulphide bridge and t         represents an integer from 1 to 15, preferably from 1 to 6 and         13         as well as the derivatives of the carboxylic acid function or         functions such as the salts, esters or amides, preferably the         amide formed with butanimide.

Within the meaning of the present invention, by “nucleofuge” is meant any ion or substituent that is able to break away from a molecule.

The carbocyclic or heterocyclic aryl radical that A can represent is preferably selected from the phenyl, naphthyl, pyridyl, thiazolyl, imidazolyl, indolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, quinolinyl, pyrazinyl, pyrrolyl, isothiazolyl radicals, and which is optionally substituted with one or more C₁ to C₆ alkyl, C₁ to C₆ alkoxy, halogen, amino or oxo groups, the preferred radical being dioxo-pyrrolyl.

The carbocyclic or heterocyclic cycloalkyl radical that A can represent is preferably selected from the cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl radicals or a heterocyclic cycloalkyl, which can be monocyclic or condensed, for example pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiazinyl.

The halogen atom is selected from fluorine, chlorine, bromine and iodine, preferably iodine.

The salts are selected from the group comprising the alkaline salts, in particular of sodium or potassium, the lithium, magnesium, calcium and barium salts, the ammonium salts, the salts of amines, the salts of amino alcohols such as triethanolamine or monoethanolamine.

The carboxylic acid esters are preferably formed with the linear or branched alkyls having from 1 to 6 carbon atoms, preferably tert-butyl.

In particular embodiments of the invention, the product corresponding to formula (I) is composed of an element X that represents a halogen, and an element A represented by an aryl radical, or an element X represented by a halogen and an element A represented by a cycloalkyl, or an element X represented by a halogen and an element A represented by a

group, or an element X represented by a halogen and an element A represented by a

group, or an element X represented by a halogen and an element A represented by a

group, or an element X represented by a nucleofuge and an element A represented by an aryl radical, or an element X represented by a nucleofuge and an element A represented by a cycloalkyl, or an element X represented by a nucleofuge and an element A represented by a

group, or an element X represented by a nucleofuge and an element A represented by a

group, or an element X represented by a nucleofuge and an element A represented by a

group, or an element X represented by a

group and an element A represented by an aryl radical, or an element X represented by a

group and an element A represented by a cycloalkyl, or an element X represented by a

group and an element A represented by a

group, or an element X represented by a

group and an element A represented by a

group, or an element X represented by a

group and an element A represented by a

group, or an element X represented by a

group and an element A represented by an aryl radical, or an element X represented by a

group and an element A represented by a cycloalkyl, or an element X represented by a

group and an element A represented by a

group, or an element X represented by a

group and an element A represented by a

group, or an element X represented by a

group and an element A represented by a

group, X₁ being a C═O or a single bond X₂ being an NH group or a single bond X₃ being an oxygen or a single bond s is equal to 1, 2 or 3 r being equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0, 1, 2 or 3; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3 or 4, W represents

-   -   an —OH radical,     -   an -L radical which represents a linker body comprising a         terminal reactive function,     -   an -FL fluorophore group used in diagnostics or analysis,         involving detection of fluorescence preferably selected from         rhodamine or a derivative thereof, preferably rhodamine B,         fluorescein isothiocyanate (FITC), a Cy dye preferably selected         from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected         from Alexa Fluor 647, 700 or 750, Texas Red, allophycocyanin         (APC) and conjugates thereof with other fluorochromes in         particular those mentioned above, phycoerythrin (PE) and         conjugates thereof with other fluorochromes in particular those         mentioned above,     -   an R* radioactive radical, which preferably contains         either a bifunctional ligand, optionally bimodal, preferably         selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA,         NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA;         or a bifunctional ligand preferably selected from one of the         acyclic chelating agents from the list constituted by EDTA,         CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA,         BOPA;         or a bifunctional ligand preferably selected from one of the         macrocyclic chelating agents from the list constituted by DOTA,         TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP,         DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide,         p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA,         NOTA, CHX-A″-DTPA;         or a bifunctional chelating agent of the cationic, anionic,         neutral or cleavable type;     -   or a radionuclide preferably selected from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y,         ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,         ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁷Au,         ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm, ⁴⁴Sc, ⁴⁷Sc,         ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb, ²¹²Bi, ²¹³Bi,         ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd, ¹⁴⁸Nd, ⁸⁹Sr,         ²¹¹At, an -L-M radical in which L has the meaning given above         and M represents a cytotoxic drug selected from a         chemotherapeutic agent or a toxin,         P is a protein comprising at least one disulphide bridge and t         represents an integer from 1 to 15, preferably from 1 to 6 and         13         as well as the derivatives of the carboxylic acid function or         functions such as the salts, esters or amides, preferably the         amide formed with butanimide.

Advantageously, the invention relates to a product characterized in that it corresponds to formula I selected from the formulae IB, IB1, IB2 and IA:

in formulae IA and IB:

X is Br or I, or a

group, or a

group, X_(a) being a halogen or a nucleofuge.

A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic,

or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3 n₄ is an integer equal to 1, 2, 3 or 4 W represents

-   -   an -L radical which represents a linker body comprising a         terminal reactive function,     -   an -FL fluorophore group used in diagnostics or analysis,         involving detection of fluorescence preferably selected from         rhodamine or a derivative thereof, preferably rhodamine B,         fluorescein isothiocyanate (FITC), a Cy dye preferably selected         from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected         from Alexa Fluor 647, 700 or 750, Texas Red, Nile Blue A,         allophycocyanin (APC) and conjugates thereof with other         fluorochromes in particular those mentioned above, phycoerythrin         (PE) and conjugates thereof with other fluorochromes in         particular those mentioned above,     -   an R* radioactive radical which preferably contains either a         bifunctional ligand, optionally bimodal, preferably selected         from the derivatives of DOTA, DTPA, C-DOTA, NODAGA, NETA,         C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA,     -   or a bifunctional ligand preferably selected from one of the         acyclic chelating agents from the list constituted by EDTA,         CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA,         BOPA,     -   or a bifunctional ligand preferably selected from one of the         macrocyclic chelating agents from the list constituted by DOTA,         TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP,         DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide,         p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA,         NOTA, CHX-A″-DTPA,     -   or a bifunctional chelating agent of the cationic, anionic,         neutral or cleavable type, or a radionuclide preferably selected         from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,         ¹⁸⁶Re, ¹⁸⁸Re, ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re,         ¹⁸⁸Re, ¹⁹⁷Au, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm,         ⁴⁴Sc, ⁴⁷Sc, ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb,         ²¹²Bi, ²¹³Bi, ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd,         ¹⁴⁸Nd, ⁸⁹Sr, ²¹¹At,     -   a cytotoxic drug M selected from a chemotherapeutic agent or a         toxin, in particular monomethyl auristatin E or monomethyl         auristatin F,     -   an -L-M radical in which L and M have the meanings given above,         P is a protein comprising at least one disulphide bridge and t         represents an integer from 1 to 15, preferably from 1 to 6 and         13         as well as the derivatives of the carboxylic acid function or         functions such as the salts, esters or amides, preferably the         amide formed with butanimide.

In a particular embodiment of the invention, at least one of the elements X1, X2 or X3 is not a single bond.

In a particular embodiment of the invention, the element X is Br or I.

In a particular embodiment of the invention, the product characterized in that it corresponds to formula (I) above has at its end a group corresponding to the following formula:

which is selected from the following radicals:

and in that the

group is selected from the following radicals:

radicals in which X is a halogen, or a nucleofuge, in the triad of atoms (T,Y,Z) each of these atoms represents, independently of the other two atoms, a carbon or nitrogen atom, and Het represents a carbocyclic or heterocyclic aryl radical or a heterocyclic cycloalkyl radical and CA represents a cycloalkyl radical.

The three atoms T, Y and Z can correspond to the triad of atoms (C,C,C) or (N,C,C) or (C,N,C) or (C,C,N) or (C,N,N) or (N,C,N) or (N,N,C) or (N,N,N), preferably (C,C,C) and (N,C,C).

In a particular embodiment of the invention, the product characterized in that it corresponds to formula (I) above has at its end a group corresponding to the following formula:

-   -   which is selected from the following radicals:

-   -   and in that the

-   -   group is selected from the following radicals:

radicals in which X is Br or I, and in the triad of atoms (T,Y,Z) each of these atoms represents, independently of the other two atoms, a carbon or nitrogen atom, in particular T is a nitrogen atom and Y and Z represent CH.

In a particular embodiment of the invention, the product characterized in that it corresponds to formula (I), selected from formulae IA and IB, comprises a

group which can be represented by a —(CH₂)₅—CO₂H radical.

In another embodiment of the invention, the product corresponding to general formula (I) constituted by formulae IA and IB in which W corresponds to an element L, so as to correspond to the following general formula IA1:

or IB1:

in which:

X is Br or I, or a

group, or a

group A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3, or 4, L represents a linker body comprising a terminal reactive function; P is a protein comprising at least one disulphide bridge, and t represents an integer from 1 to 15, preferably from 1 to 6 and 13, as well as the derivatives of the carboxylic acid function or functions such as the salts, esters or amides, preferably the amide formed with butanimide.

Within the meaning of the present invention, by “terminal reactive function” is meant any atom or group of atoms allowing one or more molecular assemblies by conventional organic chemistry or combinatorial chemistry.

In another particular embodiment of the invention, the product according to the invention comprises a linker body L corresponding to general formula III, IIIa or IIIb below:

in which K represents a (AA) n₅ radical representing a sequence of n5 identical or different, natural or non-natural amino acids or K represents a cleavage site recognized by an enzyme or K represents a hydrazino radical optionally coupled to the (AA) n₅ radical, or K represents a saccharide group preferably selected from a beta-glucuronic acid or a beta-D-galactose or a beta-D-glucose or an alpha-D-mannose or an N-acetyl-D-glucosaminyl or an N-acetyl-D-galactosaminyl, a D-glucuronyl, an L-iduronyl, a D-glucopyranosyl, a D-galactopyranosyl, a D-mannopyranosyl or L-fucopyranosyl. X represents a hydrogen or an NO₂ group, A preferably represents a hydrogen atom, Y represents a

chain or a single bond or a chemical spacer arm preferably selected from the group formed by the linear or branched alkyl radicals having from 1 to 30 carbon atoms optionally interrupted by one or more oxygen, sulphur or nitrogen atoms, for example a polyethylene glycol.

Z represents a leaving group, and n₅ represents an integer from 1 to 6.

The leaving group Z can be selected from the group comprising para nitro or para cyano phenyloxy. Preferably, the leaving group Z is one of the following radicals:

or a chlorine atom.

In another embodiment of the invention, the product corresponding to general formula III, or IIIa or IIIb comprises the (AA) n₅ radical, which is such that n₅ is equal to 1, 2, 3, 4 or 5, and AA is a pair of amino acids, said amino acids being selected from the group constituted by: a valine, a citrulline, a phenylalanine, a lysine, an aspartic acid, a methionine, an asparagine, a proline, an isoleucine, an alanine, an arginine, a glycine or a glutamic acid.

In a preferred embodiment, n₅ is equal to 2 or 3, preferably 2 and (AA) n₅ is a pair of amino acids selected from: a valine and a citrulline, or a phenylalanine and a lysine, or a valine and an alanine, or a valine and an aspartic acid, or a lysine and a methionine, or a lysine and an asparagine, or a proline and an isoleucine, or a proline and a lysine, or a lysine and a methionine, or a lysine and an asparagine, or a valine and a lysine, or an alanine and a lysine, or a phenylalanine and a lysine, or two phenylalanines and a lysine, or an alanine, or a phenylalanine and a lysine, or two arginines, or a lysine and an arginine, or a glutamic acid, or a glycine and an arginine, or two glycines and an arginine. In a particular embodiment of the invention, the product corresponding to formula III, or IIIa, or IIIb, comprises an element K that represents a site of cleavage by an enzyme selected from the enzymes of the cathepsin B, cathepsin C, cathepsin D type, the enzymes selected from plasmin, a lysosomal protease, a lysosomal enzyme, or urokinase plasminogen activator (uPA), elastase, proteinase 3, cathepsin G.

K can also represent a recognition site of cleavage by an enzyme of the matrix metalloproteinase (MMP) type. In this case, the enzyme of the matrix metalloproteinase (MMP) type is preferably selected from collagenases 1, 2 and 3 (MMP-1, MMP-8, MMP-13), gelatinases A and B (MMP-2 and MMP-9), stromelysins 1 and 2 (MMP-3 and MMP-10), matrilysins 1, 2 and 3 (MMP-7, MMP-26, MMP-11), macrophage metalloelastase (MMP-12), the membrane MMPs (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25), enamelysin (MMP-20), CA-MMP (MMP-23), epilysin (MMP-18) and PSMA.

K can also represent a recognition site of cleavage by an esterase, a carboxylesterase, alkaline phosphatases, proteases, peptidases, cathepsins, glucosidase, galactosidase, β-D-galactosidase, induronidase, β-glucuronidase, mannosidase, N-acetyl-D-glucosaminidase or N-acetyl-D-galactosaminidase.

In the context of the present invention, by “cleavage site” is meant the position where cleavage of the peptide chain by an enzyme takes place, for example the Valine-Citrulline site, the above cleavage sites K or the position where enzyme recognition takes place. In a particular embodiment of the invention, the product corresponds to general formula IA2

or to formula IB2

in which X is Br or I, or a

group, or a

group A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3 or 4, and L represents a linker body comprising a terminal reactive function, P is a protein comprising at least one disulphide bridge, t represents an integer from 1 to 15, preferably from 1 to 6 and 13, M is a cytotoxic drug selected from a chemotherapeutic agent or a toxin, as well as the derivatives of the carboxylic acid function or functions such as the salts, esters or amides, preferably the amide formed with butanimide.

Within the meaning of the present application, by “cytotoxic drug” is meant any natural or synthetic molecule capable of inhibiting or preventing the function of the cells. By “cytotoxic” is also meant the property of a chemical or biological agent of altering cells, optionally until they are destroyed.

In a particular embodiment of the invention, the drug M is a drug selected from any compound for which marketing authorization has been obtained and which is used in anticancer or anti-inflammatory therapy, and any molecule undergoing clinical evaluation in anticancer or anti-inflammatory therapy. The drug M will be selected for example from paclitaxel (Taxol®) or docetaxel (Taxotere®) or a derivative thereof, topotecan, bortezomib, daunorubicin, the analogues of daunorubicin, vincristine, mitomycin C, retinoic acid, methotrexate, Ilomedin, aspirin, an IMIDs, lenalidomide, pomalidomide. In another particular embodiment of the invention, the drug M is selected from the group constituted by duocarmycin and its analogues, the dolastatins, combretastatin, calicheamicin, N-acetyl-γ-calicheamycin (CMC), a calicheamycin derivative, maytansine and its analogues, DM-I, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin, disorazole, the epothilones, echinomycin, estramustine, cemadotine, eleutherobin, methopterin, actinomycin, mitomycin A, camptothecin, a camptothecin derivative, SN38, maytansine, a derivative of the maytansinoid type, DM1, DM4, TK1, amanitin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, a pyrrolopyridodiazepine, a pyrrolopyridodiazepine dimer, a DNA intercalator, a histone deacetylase inhibitor, or a tyrosine kinase inhibitor.

In another particular embodiment of the invention, the drug M is selected from pseudomonas exotoxin (PE), deBouganin, Bouganin, diphtheria toxin (DT) and ricin.

In another particular embodiment of the invention, the product according to the invention corresponds to the following formula IB2:

in which:

X is Br or I, or a

group, or a

group A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3 or 4, L represents a linker body comprising a terminal reactive function, M is a cytotoxic drug or a toxin, P is a protein comprising at least one disulphide bridge, and t represents an integer, preferably from 1 to 6 and 13, as well as the derivatives of the carboxylic acid function or functions such as the salts, esters or amides, preferably the amide formed with butanimide.

In a particular embodiment of the invention, the protein P comprising at least one disulphide bridge is selected from an agonist or an antagonist, an antibody or an antibody fragment, a fusion protein comprising an antibody or an antibody fragment, an albumin or a peptide.

By “agonist” is meant any molecule which, on binding specifically to a given cell receptor, triggers the same effects as the natural ligand.

By “antagonist” is meant any molecule which, on binding specifically to a given cell receptor, can thus block the action of the endogenous ligand by opposing binding of the ligand to its receptor.

By “antibody”, also commonly called “immunoglobulin”, is meant a heterotetramer constituted by two heavy chains of approximately 50-70 kDa each (called the H chains, for Heavy) and two light chains of approximately 25 kDa each (called the L chains, for Light), joined together by intra- and interchain disulphide bridges.

Each chain is constituted, in the N-terminal position, by a variable region or domain, called VL for the light chain, VH for the heavy chain and, in the C-terminal position, by a constant region, constituted by a single domain called CL for the light chain and of three or four domains called CH1, CH2, CH3, CH4, for the heavy chain.

By “antibody fragment” is meant any part of an immunoglobulin obtained by enzymatic digestion or obtained by bioproduction.

The antibody according to the present invention can be a chimeric monoclonal antibody, humanized or human, monospecific or bispecific.

By “chimeric antibody” is meant an antibody in which the sequences of the variable regions of the light chains and of the heavy chains belong to a species different from that of the sequences of constant regions of the light chains and of the heavy chains.

For the purposes of the invention, the sequences of the variable regions of the heavy and light chains are preferably of murine origin whereas the sequences of the constant regions of the heavy and light chains belong to a non-murine species. In this respect, for the constant regions, all the species of non-murine mammals can be used, and in particular human, monkey, Old-World swine, bovides, equines, felids, canines or birds, this list not being exhaustive.

Preferably, the chimeric antibodies according to the invention contain sequences of constant regions of heavy and light chains of human origin and the sequences of variable regions of heavy and light chains of murine origin.

By “humanized antibody” is meant an antibody for which some or all of the sequences of the regions involved in antigen recognition (the hypervariable regions or CDR: Complementarity Determining Region) and sometimes certain amino acids of the FR regions (FR: Framework Regions)) are of non-human origin whereas the sequences of the constant regions and variable regions not involved in antigen recognition are of human origin.

By “human antibody” is meant an antibody containing only human sequences, both for the variable and constant regions of the light chains and for the variable and constant regions of the heavy chains.

In another particular embodiment of the invention, the protein P can be an antibody fragment. This antibody fragment is selected from the group constituted by: Fab, F(ab)′2, Fc, F′c, pFc′, ScFv, Fv, Fd, Fabc, diabody, minibody, ScFv-Fc or ScFv-Fv.

Enzymatic digestion of the immunoglobulins by papain generates two identical fragments, which are called the Fab (Fragment antigen binding) fragments, and one Fc (Fragment crystallizable) fragment.

Enzymatic digestion of the immunoglobulins by pepsin generates a F(ab′)2 fragment and an Fc fragment split into several peptides.

F(ab′)2 is formed by two Fab′ fragments linked by interchain disulphide bridges.

The Fab parts are constituted by the variable regions and the CH1 and CL domains, whereas the Fc region is constituted by the two globular domains CH2 and CH3 (and CH4 when it is present).

The Fab′ fragment is constituted by the Fab region and a hinge region. Fab′-SH refers to a Fab′ fragment in which the cysteine residue of the hinge region bears a free thiol group (Carter et al., Nature BioTechnology 10: 163-167 (1992)). The Fv fragment is composed of the domains VH and VL linked by disulphide bridges. It is the smallest fragment retaining antigen binding activity.

The Fd fragment is formed by the VH and CH1 domains.

The “F′c”, “pFc′”, and “Fabc” fragments are defined in FIGS. 5A to 5C.

The scFv (single chain Fragment variable) is a fragment originating from protein engineering which is constituted only by the variable domains VH and VL. The structure is stabilized by a short flexible peptide arm, called a linker, which is placed between the two domains.

The ScFv fragment can be bound to an Fc fragment in order in order to produce an ScFv-Fc or to an Fv fragment in order in order to produce an ScFv-Fv.

If the size of the binding peptide is reduced, new steric constraints appear between the scFvs; the VH and VL domains can no longer combine into a functional structure and multimeric structures are obtained (dimeric: “diabody”, trimeric: “triabody” and tetrameric: “tetrabody”).

In the context of the present invention, diabodies can be used. They can have multiple valences and specificities.

The term “valent” denotes the presence of a defined number of antigen binding sites in an antibody molecule.

The term “specific” refers to the different types of antigens that can bind to the same antibody.

Thus, in the present invention, by “diabody” is meant an scFv dimer, said diabody being divalent, mono- or bispecific according to whether it binds two identical or different antigens.

The Fab, Fv and scFv fragments are monovalent and monospecific.

By “minibody” is meant a fragment composed of a light chain, of a heavy chain bound to a CH3 group.

In the context of the present invention, the antibody or antibody fragment is directed against a tumour antigen or inflammation antigen.

In a particular embodiment of the invention, the antibody or antibody fragment is directed against one of the antigens of the cluster of differentiation (CD), the identification number of which varies between CD1a and CD363; in this list, the following CDs are preferred: CD1a, CD363, CD3, CD4, CD13, CD19, CD20, CD21, CD22, CD25, CD30, CD31, CD33, CD34, CD37, CD39, CD40, CD44, CD47, CD52, CD56, CD66e, CD70, CD72, CD73, CD74, CD79, CD79b, CD80, CD86, CD117, CD138, CD194, CD205, CD227 or CD248. The antibody or antibody fragment can also be directed against one of the antigens selected from the list formed by the following: CA125, G250, GD2, HLA-DRβ, MUC1, VEGF, VEGFA, VEGF-R1/2, TRAIL-R2 (DR5), EpCAM, GPIIb, GPIIIa, TNF alpha, TNFR, TNT, Lewis Y, EGFR, HER-2, HER-3, HER-3 MM-111, HER-4, homodimer or heterodimer between members of the erbbn family (n between 1 and 4), AXL, Protein F, IgE-Fc, VEGF-A, integrin α4, integrin α4β7, integrin αV, C5, IL-6R, IL-6Rα, IL12, IL15, IL18, IL23, IL-1β, IL-1, TPO-R, GPNMB, PSMA, PSA, PAP, PSM, integrin αv, Cripto, TACSTD2 (TROP2 or EGP1), CEA, Folate receptor 1, Mucin 16, Endothelin Receptor ETB, STEAP1, SLC44A4 (AGS-5), Nectin 4, AGS-16, Guanylyl cyclase C, Mucin 1, EGFRvIII, Mesothelin, IL2R, A33, Can, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGFR-1, VEGFR-2, VEGFR-3, TGFP, TGFPR, FGF, FGF8b, FGFR, PDGF, PDGFR, PDGFRα, PDGFRβ, Ang-1, Ang-2, integrin, αvβ3, αvβ5, α3β1, α6β4, α2β1, anti-integrin α4, RANK-L, BLyS, c-MET, DR, DR10, TCRαβ, ICOS, CTLA-4, CAIX (MN), EphA2, CA6, ovarian CA6, cervical CA6, breast CA6, angiopoietin-2, Cripto, ENPP3, Mesothelin, FOLR1, Nectin-4, TIM-1, Muc-16, Tissue Factor, LIV-1, GM2, α5 integrin, TLR-7, PD-1, AFP, CA125 (MUC16), Sialyl LewisY, CAMPATH-1, HLA-DR, anti-idiotype, carcinoembryonic antigen (CEA), TAG-72, Folate-binding protein, A33, G250, gangliosides (including GD2, GD3, GM2), LeY, collagen 4 (collagen IV), collagen 18 (collagen XVIII), SC6, CA-125, CA19-9, p185HER2, de2-7 EGFR, Fibroblast activation protein (FAP), Tenascin, metalloproteinases, Endosialin, Carbonic anhydrase, Galectin 9, Aldolase A, eIFγ4, Tyrosinase, Galectin 4, HERKV-K10, p53, NY-LU-12, Restin, NY-CO-38, MAGE-1, MAGE-4a, SSX2, NY-ESO-1, SCP-1, HGFR, PTK 7, CCK-4, PDGFR, PTP-LAR, CDCP1, CADM1, IGSF4, Lu, BCAM, CEACAM6, JAM-A, PTGFRN (CD9P-1), MCAM, MUC18, MCP, EMMPRIN, TfR, TRAILR2, ClqR, hTERT, Survivin, MDM2, CYP1B1, Melan-A, MART-1, MART-2, Melanosomal proteins, gp100, neo-PAP, CDC27, MAGEs, WT1, MUM-1, MUM-2, MUM-3, BRAF, TPI, fibronectin, K-ras, β-catenin, CDK4, caspase-8, p14ARF, p16INK4a, TGFβRII, bcr-abl, SYT-SSX, TRP-1, TRP-2, GnT-V, tyrosinase, FGF5, TEL-AML1, Proteinase 3, HER2/neu, AFP, MUC-1, EBV-EBNA, HTLV-1 tax, HPV16-E7, mutated HLA-A2, HAL SART3, GnT-V, CEACAM5, AGS-16, GPNMB, ESAT-6, RANK, CanAg, fibrin, TF, PRAME, CA19-9, CA50, CA125, CA195, CAM17.1/WGA, AFP, β2-MG, DU-PAN2, HE4, b-2 microglobulin, transferrin, transthyretin, ApoA1, TROP-2, CTLA-4, GITR, PD-1, PD-L1, c-KIT, CD11b-CD18 integrin heterodimer, DNA/Histon H1, Folate, EpCAM, Tenascin-c, ECM (proteoglycan or fibronectin), fibrinogen, SV40 large T antigen, SC6-Ag, SC-Ag, DR4 (death receptor 4), DR5, ESA, mucin, hPAM4, hRS7, HLA-DR, CCR4, MTX1, MTX2, PECAM, thrombomodulin Tn, cathepsin D, TYRO-3, MER. The antibody or antibody fragment can also be directed against a PF4/heparin complex. The invention also relates to a method for preparing the products of formulae I, characterized in that, if desired, a product of formula IA3

as well as the derivatives of the carboxylic acid function or functions such as the salts, esters or amides, preferably the amide formed with butanimide, where in formula IA3:

X is Br or I, or a

group, or a

group, X_(a) being a halogen or a nucleofuge A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond) X₂ is an NH group or a C═O or a single bond) X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3, or 4, is reacted with a product of formula H2N-L, or HO-L in which L represents a linker body comprising a terminal reactive function, in order to obtain a product of formula IA1:

and, if desired, the product of formula IA1 is reacted with a product of formula: H2N-M, or HO-M in which M represents a cytotoxic drug selected from a chemotherapeutic agent or a toxin, in order to obtain a product of formula IA2:

or product of formula IA3 which, if desired, is reacted with a residue of an -FL fluorescent radical in order to obtain a product of formula (IA4)

or product of formula IA3 which, if desired, is reacted with a residue of an —R* radioactive radical in order to obtain a product of formula (IA5)

The products of formulae IA1 to IA5 corresponding to the products of formula IA which, if desired, are reacted with a product of formula P, P being a protein comprising at least one disulphide bridge and t represents an integer from 1 to 15, preferably from 1 to 6 and 13, in order to obtain a product of formula (IB):

in which W represents

-   -   an -L radical which represents a linker body comprising a         terminal reactive function,     -   an -FL fluorophore group used in diagnostics or analysis,         involving detection of fluorescence preferably selected from         rhodamine or a derivative thereof, preferably rhodamine B,         fluorescein isothiocyanate (FITC), a Cy dye preferably selected         from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected         from Alexa Fluor 647, 700 or 750, Texas Red, Nile Blue A,         allophycocyanin (APC) and conjugates thereof with other         fluorochromes in particular those mentioned above, phycoerythrin         (PE) and conjugates thereof with other fluorochromes in         particular those mentioned above,     -   an R* radioactive radical which preferably contains     -   either a bifunctional ligand, optionally bimodal, preferably         selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA,         NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA,     -   or a bifunctional ligand preferably selected from one of the         acyclic chelating agents from the list constituted by EDTA,         CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA,         BOPA,     -   or a bifunctional ligand preferably selected from one of the         macrocyclic chelating agents from the list constituted by DOTA,         TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP,         DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide,         p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA,         NOTA, CHX-A″-DTPA,     -   or a bifunctional chelating agent of the cationic, anionic,         neutral or cleavable type, or a radionuclide preferably selected         from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,         ¹⁸⁶Re, ¹⁸⁸Re, ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re,         ¹⁸⁸Re, ¹⁹⁷Au, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm,         ⁴⁴Sc, ⁴⁷Sc, ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb,         ²¹²Bi, ²¹³Bi, ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd,         ¹⁴⁸Nd, ⁸⁹Sr, ²¹¹At,         -   a cytotoxic drug M selected from a chemotherapeutic agent or             a toxin, in particular monomethyl auristatin E or monomethyl             auristatin F,         -   an -L-M radical, in which L and M have the meanings given             above.

In a particular embodiment of the invention, the products of formula IA1 to IA5 of the method of preparation according to the invention are attached to the protein P on at least one of its disulphide bridges by a reaction of nucleophilic substitution, typically in a phosphate buffer, in the presence of TCEP.

In a particular embodiment of the invention, the products of formula IB and in particular the products of formula IB2 are used in the treatment of a tumour, in particular colorectal cancer, hepatocarcinoma, a lung cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, colorectal cancer, K-RAS mutated colorectal cancer, a breast cancer, triple-negative breast cancer, metastatic breast cancer, liver cancer, head and neck cancer, Castleman disease, thyroid cancer, medulloblastoma, glioblastoma multiforme, glioma, sarcoma, anaplastic astrocytoma, kidney cancer, stomach cancer, malignant ascites, prostate cancer (metastatic or not), solid tumours, acute myeloid leukaemia, chronic lymphocytic leukaemia, melanoma, myeloma, multiple myeloma, lymphomas, mantle cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma.

In another particular embodiment of the invention, the products of formula IB and in particular the products of formula IB2 are used in the treatment of an inflammatory or autoimmune disease in particular graft rejection, rheumatoid arthritis, Crohn's disease, psoriasis, multiple sclerosis, cryopyrin-associated periodic syndromes, thrombocytopenic purpura.

In another embodiment of the invention, the products of formula I, constituted by formulae IA:

and IB:

in which:

X is Br or I, or a

group, or a

group, X_(a) being a halogen or a nucleofuge A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3 or 4, W represents

-   -   an -L radical which represents a linker body comprising a         terminal reactive function,     -   an -FL fluorophore group used in diagnostics or analysis,         involving detection of fluorescence preferably selected from         rhodamine or a derivative thereof, preferably rhodamine B,         fluorescein isothiocyanate (FITC), a Cy dye preferably selected         from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected         from Alexa Fluor 647, 700 or 750, Texas Red, Nile Blue A,         allophycocyanin (APC) and conjugates thereof with other         fluorochromes in particular those mentioned above, phycoerythrin         (PE) and conjugates thereof with other fluorochromes in         particular those mentioned above,     -   an R* radioactive radical which preferably contains     -   either a bifunctional ligand, optionally bimodal, preferably         selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA,         NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA,     -   or a bifunctional ligand preferably selected from one of the         acyclic chelating agents from the list constituted by EDTA,         CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA,         BOPA,     -   or a bifunctional ligand preferably selected from one of the         macrocyclic chelating agents from the list constituted by DOTA,         TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP,         DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide,         p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA,         NOTA, CHX-A″-DTPA,     -   or a bifunctional chelating agent of the cationic, anionic,         neutral or cleavable type, or a radionuclide preferably selected         from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,         ¹⁸⁶Re, ¹⁸⁸Re, ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re,         ¹⁸⁸Re, ¹⁹⁷Au, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm,         ⁴⁴Sc, ⁴⁷Sc, ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb,         ²¹²Bi, ²¹³Bi, ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd,         ¹⁴⁸Nd, ⁸⁹Sr, ²¹¹At,         -   a cytotoxic drug M selected from a chemotherapeutic agent or             a toxin, in particular monomethyl auristatin E or monomethyl             auristatin F,         -   an -L-M radical in which L and M have the meanings given             above,             P is a protein comprising at least one disulphide bridge,             and t represents an integer, preferably from 1 to 6 and 13,             are used in diagnostics or in an analysis involving             detection of fluorescence.

In another particular embodiment of the invention, the products of formula IA4:

or of formula IB4

(IB4)

in which:

X is Br or I, or a

group, or a

group, X_(a) being a halogen or a nucleofuge A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3 or 4, W represents

-   -   an -L radical which represents a linker body comprising a         terminal reactive function,     -   an -FL fluorophore group used in diagnostics or analysis,         involving detection of fluorescence preferably selected from         rhodamine or a derivative thereof, preferably rhodamine B,         fluorescein isothiocyanate (FITC), a Cy dye preferably selected         from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected         from Alexa Fluor 647, 700 or 750, Texas Red, Nile Blue A,         allophycocyanin (APC) and conjugates thereof with other         fluorochromes in particular those mentioned above, phycoerythrin         (PE) and conjugates thereof with other fluorochromes in         particular those mentioned above,     -   an R* radioactive radical which preferably contains     -   either a bifunctional ligand, optionally bimodal, preferably         selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA,         NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA,     -   or a bifunctional ligand preferably selected from one of the         acyclic chelating agents from the list constituted by EDTA,         CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA,         BOPA,     -   or a bifunctional ligand preferably selected from one of the         macrocyclic chelating agents from the list constituted by DOTA,         TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP,         DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide,         p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA,         NOTA, CHX-A″-DTPA,     -   or a bifunctional chelating agent of the cationic, anionic,         neutral or cleavable type,     -   or a radionuclide preferably selected from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y,         ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,         ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁷Au,         ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm, ⁴⁴Sc, ⁴⁷Sc,         ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb, ²¹²Bi, ²¹³Bi,         ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd, ¹⁴⁸Nd, ⁸⁹Sr,         ²¹¹At,         -   a cytotoxic drug M selected from a chemotherapeutic agent or             a toxin, in particular monomethyl auristatin E or monomethyl             auristatin F,         -   an -L-M radical in which L and M have the meanings given             above,             P is a protein comprising at least one disulphide bridge,             and t represents an integer, preferably from 1 to 6 and 13,             correspond to the products of formula IA and IB in which W             represents an -FL fluorescent radical, are used in             diagnostics or in analysis involving detection of             fluorescence. In this case, FL is a fluorophore (or             fluorochrome) preferably selected from rhodamine or a             derivative thereof (preferably rhodamine B), fluorescein             isothiocyanate (FITC), a Cy Dye (preferably selected from             Cy5, Cy5.5, Cy7), an Alexa fluor dye (preferably selected             from Alexa Fluor 647, 700 or 750), Texas Red, Nile Blue A,             allophycocyanin (APC) and conjugates thereof with other             fluorochromes in particular those mentioned above,             phycoerythrin (PE) and conjugates thereof with other             fluorochromes in particular those mentioned above, or any             fluorophore used in diagnostics or analysis, involving             detection of fluorescence.

The use of radioactive elements is very widespread in the medical field, whether for the needs of imaging or of therapy as illustrated by the article by Cutler et al., Chemical Reviews, 2013, 113, 858-883.

In yet another particular embodiment of the invention, the products of formula (IA5):

or of formula (IBS):

in which X is Br or I, or a

group, or a

group, X_(a) being a halogen or a nucleofuge A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3 or 4, and R* is a radioactive radical, P is a protein comprising at least one disulphide bridge, and t represents an integer, preferably from 1 to 6 and 13 are used in diagnostics or in analysis involving detection of radioactivity. R* contains a bifunctional ligand, optionally bimodal, preferably selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA, NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA. R* contains a bifunctional ligand preferably selected from one of the following acyclic chelating agents: EDTA, CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy2DTPA, DTPA-MA, DTPA-BA, BOPA. R* contains a bifunctional ligand preferably selected from one of the following macrocyclic chelating agents: DOTA, TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP, DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide, p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA, NOTA, CHX-A″-DTPA. R* contains a bifunctional chelating agent of the cationic, anionic, neutral or cleavable type. R* contains a radionuclide preferably selected from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁷Au, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm, ⁴⁴Sc, ⁴⁷Sc, ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd, ¹⁴⁸Nd, ⁸⁹Sr, ²¹¹At,

The products of formula (IA5) and (IB5) can also be used for non-invasive imaging techniques, used in diagnostics for measuring distribution and function in a biological system, of the PET (positron emission tomography) or SPECT (single photon emission computed tomography) type.

The products of formula (IA5) and (IB5) can also be used as radiopharmaceuticals, for example for treating a cancer.

In a particular embodiment of the invention, the products of formula (IA5) and (IB5) are used in non-invasive imaging techniques, used in diagnostics for measuring distribution and function in a biological system, of the PET (positron emission tomography) or SPECT (single photon emission computed tomography) type.

In another particular embodiment of the invention, the products of formula (IA5) and (IB5) are used as radiopharmaceuticals, for example for treating a cancer.

The invention also relates to a method for preparing the products of formula IB as defined above, characterized in that a product of formula IA

where in formula (IA)

X is Br or I, or a

group, or a

group, X_(a) being a halogen or a nucleofuge. A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

group, a

group or a

group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3, n₄ is an integer equal to 1, 2, 3 or 4, W represents

-   -   an -L radical which represents a linker body comprising a         terminal reactive function,     -   an -FL fluorophore group used in diagnostics or analysis,         involving detection of fluorescence preferably selected from         rhodamine or a derivative thereof, preferably rhodamine B,         fluorescein isothiocyanate (FITC), a Cy dye preferably selected         from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected         from Alexa Fluor 647, 700 or 750, Texas Red, Nile Blue A,         allophycocyanin (APC) and conjugates thereof with other         fluorochromes in particular those mentioned above, phycoerythrin         (PE) and conjugates thereof with other fluorochromes in         particular those mentioned above,     -   an R* radioactive radical, which preferably contains         either a bifunctional ligand, optionally bimodal, preferably         selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA,         NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA;         or a bifunctional ligand preferably selected from one of the         acyclic chelating agents from the list constituted by EDTA,         CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA,         BOPA;         or a bifunctional ligand preferably selected from one of the         macrocyclic chelating agents from the list constituted by DOTA,         TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP,         DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide,         p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA,         NOTA, CHX-A″-DTPA;         or a bifunctional chelating agent of the cationic, anionic,         neutral or cleavable type;         or a radionuclide preferably selected from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y,         ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,         ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁷Au,         ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm, ⁴⁴Sc, ⁴⁷Sc,         ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb, ²¹²Bi, ²¹³Bi,         ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd, ¹⁴⁸Nd, ⁸⁹Sr,         ²¹¹At,     -   a cytotoxic drug M selected from a chemotherapeutic agent or a         toxin, in particular monomethyl auristatin E or monomethyl         auristatin F,     -   an -L-M radical in which L and M have the meanings given above,         in particular the

-   -   group is selected from the following radicals:

as well as the derivatives of the carboxylic acid function or functions such as the salts, esters or amides, preferably the amide formed with butanimide, is reacted with a product of formula P, in which P is a protein comprising at least one disulphide bridge, in order to obtain a product of formula (IB):

in which t represents an integer from 1 to 15, preferably from 1 to 6 and 13.

DESCRIPTION OF THE FIGURES

FIG. 1 presents the general structure of an antibody-drug conjugate. The conventional modification sites are as follows:

4 lysines

Average of 4 residues modified among the 8 residues accessible for modification from among 80, consequently numerous DAR species.

4 disulphide bridges

After reduction with TCEP (tris(2-carboxyethyl)phosphine), 8 cysteine residues accessible, consequently numerous species with DAR from 0 to 8.

DAR=drug-antibody ratio (or drug-to-antibody ratio).

The average DAR is approximately 4 with conventional modifications.

FIG. 2 presents the MALDI-TOF mass spectrometry analysis of trastuzumab (peak at 74095 and peaks indicated by the dotted arrows) and of grafted trastuzumab reconstructed by the linker (spacer) 6-(3,4-dibromomaleimido)hexanoic acid (peak at 74846 and peaks indicated by the solid arrows).

This analysis is able to confirm that the reaction mixture (peak at 74095) contains 89% of the species resulting from grafting 4 linkers of 6-(3,4-dibromomaleimido)hexanoic acid on the antibody, the grafting average being 4.0:

Calculation of the distribution of the different species by deconvolution of the main peak (peak at 74846):

FIG. 3 presents the HIC-HPLC chromatogram of the trastuzumab antibody 24 purified and reconstructed after reduction by TCEP and then addition of the linker 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-OH.

A small quantity of DAR=0 remains (confirmation of the MALDI-TOF results) and DAR=4 is the main species.

FIGS. 4A to 4C present the nomenclature of the different antibody fractions.

FIG. 4A presents the Igl fragments obtained after digestion with papain (Fc and F′c) or pepsin (Fc′=pepF′c).

FIG. 4B presents the Fabc, Fd, Fv and scFv fragments.

FIG. 4C presents the structure of the chimeric and humanized antibodies.

FIG. 5 presents the RP-HPLC chromatogram of the trastuzumab-(maldiBr-linker7)₄ antibody 24 obtained in example 5.2.

A: trastuzumab.

B: antibody reduced by TCEP: the heavy and light chains of trastuzumab are separated due to the denaturing nature of RP-HPLC analysis.

C: reconstructed trastuzumab 24 (crude reaction mixture): a single peak is observed, proof that the bridges between the chains have been reconstructed by the linker 7 6-(3,4-dibromomaleimido)hexanoic acid.

The peaks where the retention times are less than 10 minutes represent the linker residues.

FIG. 6 presents the RP-HPLC analysis of trastuzumab-(maldiBr-linker8)₄ 25 obtained in example 5.3.

A: native trastuzumab.

B: trastuzumab reduced by TCEP.

C: trastuzumab 25 reconstructed by the linker 8 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-OH.

FIG. 7 presents the RP-HPLC analysis of trastuzumab-(maldiSPh-linker26)₄ 27 obtained in example 5.4.

A: native trastuzumab.

B: trastuzumab reduced by TCEP.

C: trastuzumab 27 reconstructed by the linker 26 6-(3,4-dithiophenylmaleimido)hexanamide-Val-Cit-PAB-OH.

FIG. 8 presents the RP-HPLC analysis of trastuzumab-(PydiMediBr-linker20)₄ 28 obtained in example 5.5.

A: native trastuzumab.

B: trastuzumab reduced by TCEP.

C: trastuzumab 28 by the linker 20 6-(2,6-bis(bromomethyl)isonicotinamido)hexanoic acid.

FIG. 9 presents the RP-HPLC analysis of trastuzumab-(PydiMediBr-linker22)₄ 29 obtained in example 5.6.

A: native trastuzumab.

B: trastuzumab reduced by TCEP.

C: trastuzumab 29 by the acid linker 22 6-(2,6-bis(bromomethyl)isonicotinamido)hexanamide-Val-Cit-PAB-OH.

FIG. 10 presents the RP-HPLC analysis of trastuzumab-(PhdiMediBr-linker15)₄ 30 obtained in example 5.7.

A: native trastuzumab.

B: trastuzumab reduced by TCEP.

C: Trastuzamab 30 partially reconstructed by the acid linker 15 6-(2,6-bis(bromomethyl)benzamido)hexanamide-Val-Cit-PAB-OH.

FIG. 11 presents the RP-HPLC analysis of rituximab-(maldiBr-linker7)₄ 31 obtained in example 5.8.

A: native rituximab.

B: rituximab reduced by TCEP.

C: rituximab 31 reconstructed by the linker 7 6-(3,4-dibromomaleimido)hexanoic acid.

FIG. 12 presents the RP-HPLC analysis of trastuzumab-(maldiBr-linker9-MMAE)₄ 32 obtained in example 5.9.

A: native trastuzumab.

B: trastuzumab reduced by TCEP.

C: trastuzumab 32 reconstructed by the linker 9-MMAE dibromomaleimido)hexanamide-Val-Cit-PAB-MMAE.

EXAMPLES Example 1 Valine-Citrulline-Alcohol Para-Aminobenzyl Group

1.1. Synthesis of Fmoc-Val-OH (1)

Valine (1.09 g, 9.31 mmol, 1 eq) is dissolved in 20 mL of water, and then 20 mL of dioxane and NaHCO₃ (1.56 g, 18.62 mmol, 2 eq) are added. The medium is cooled to 0° C., then Fmoc-Cl (2.65 g, 10.24 mmol, 1.1 eq) is introduced and the reaction is left for 1 h at 0° C. and then stirred overnight at ambient temperature. The next day, the reaction medium is acidified to pH=2 with 1M HCl solution and then extracted with EtOAc (3×60 mL). After evaporation under vacuum, the yellow oil obtained is purified by flash chromatography from 0 to 40% of EtOAc in cyclohexane. A white solid is obtained (2.23 g, 71%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.78-7.29 (m, 8H; Ar Fmoc), 5.25 (d, J=9.0 Hz, 1H; CONH Val), 4.43 (d, J=7.0 Hz, 2H; CH₂ Fmoc), 4.37 (dd, J=4.5 Hz, J=9.0 Hz, 1H; H_(α) Val), 4.24 (t, J=7.0 Hz, 1H; CH Fmoc), 2.29-2.23 (m, 1H; H_(β) Val), 1.02, 0.96 (2d, J=7.0 Hz, 6H; CH₃ Val)

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 176.50 (1C, COOH), 156.48 (1C, C═O Fmoc), 143.85, 141.47 (4C, C—Ar Fmoc), 127.89, 127.23, 125.21, 120.16 (8C, CH—Ar Fmoc), 67.27 (1C, CH₂ Fmoc), 58.93 (1C, C_(α) Val), 47.32 (1C, CH Fmoc), 31.15 (1C, C_(β) Val), 19.17, 17.59 (2C, CH₃ Val)

1.2. Synthesis of Fmoc-Val-OSu (2)

Fmoc-Val-OH (735 mg, 2.17 mmol, 1 eq) and N-hydroxysuccinimide (375 mg, 3.26 mmol, 1.5 eq) are dissolved in 5.5 mL of THF. DCC (448 mg, 2.17 mmol, 1 eq) is added at 0° C. and the reaction is stirred for 2 h at this temperature. The medium is then filtered on a frit and the solid is washed with THF (3×3 mL). The organic phase is dried over MgSO₄ and evaporated under vacuum and then purified by flash chromatography from 0 to 40% of EtOAc in cyclohexane in order to produce a colourless gum (865 mg, 91%).

HRMS: m/z calculated: 437.10671 [M+H]+. found: 437.17074.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.78-7.29 (m, 8H; Ar Fmoc), 5.28 (d, J=9.0 Hz, 1H; CONH Val), 4.69 (dd, J=5.0 Hz, J=9.0 Hz, 1H; H_(α) Val), 4.45 (d, J=7.0 Hz, 2H; CH₂ Fmoc), 4.24 (t, J=7.0 Hz, 1H; CH Fmoc), 2.84 (s, 4H; NCOCH₂), 2.40-2.29 (m, 1H; H_(β) Val), 1.07, 1.05 (2d, J=7.0 Hz, 6H; CH₃ Val)

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 168.72, 167.87 (3C, C═O Su, C═O Val), 155.97 (1C, C═O Fmoc), 143.94, 143.72, 141.42 (4C, C—Ar Fmoc), 127.85, 127.23, 125.17, 120.10 (8C, CH—Ar Fmoc), 67.35 (1C, CH₂ Fmoc), 57.60 (1C, C_(α) Val), 47.25 (1C, CH Fmoc), 31.75 (1C, C_(β) Val), 25.69 (2C, CH₂C(O)NO), 18.82, 17.41 (2C, CH₃ Val)

1.3. Synthesis of Fmoc-Val-Cit-OH (3)

L-Citrulline (84 mg, 0.481 mmol, 1.05 eq) and NaHCO₃ (40 mg, 0.481 mmol, 1.05 eq) are dissolved in 1.2 mL of water and a solution of Fmoc-Val-OSu (200 mg, 0.458 mmol, 1 eq) in 1.2 mL of DME is added. To improve the solubility, 1.2 mL of THF is added and the medium is stirred for 21 h. 2.3 mL of 15% citric acid is added and the medium precipitates; it is extracted with a 10% iProH/EtOAc mixture (3×3 mL). The combined organic phases are washed with NaCl solution (2×4.5 mL), dried over MgSO₄ and then evaporated. When the white solid obtained is properly dry, it is washed with ether in order to produce an electrostatic white solid (140 mg, 62%).

HRMS: m/z calculated: 497.26142 [M+H]+. found: 497.23945.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 12.50 (br s, 1H; COOH), 8.17 (d, J=7.5 Hz, 1H; CONH Cit), 7.88-7.27 (m, 9H; Ar Fmoc, CONH Val), 5.91 (t, J=5.5 Hz, 1H; NH Cit), 5.36 (s, 2H; NH₂ Cit), 4.25-4.07 (m, 4H; CH₂ Fmoc, CH Fmoc, H_(α) Cit), 3.89 (dd, J=7.0 Hz, J=9.0 Hz, 1H; H_(α) Val), 2.94-2.88 (m, 2H; NCH₂ Cit), 1.97-1.88 (m, 1H; H_(β) Val), 1.70-1.31 (m, 4H; CH₂ Cit), 0.86, 0.82 (2d, J=7.0 Hz, 6H; CH₃ Val)

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 173.45, 171.31 (2C, COOH, C═O Val), 158.76 (1C, NH₂CO Cit), 156.07 (1C, C═O Fmoc), 143.95, 143.80, 140.72 (4C, C—Ar Fmoc), 127.67, 127.09, 125.43, 120.11 (8C, CH—Ar Fmoc), 65.69 (1C, CH₂ Fmoc), 59.83 (1C, C_(α) Val), 51.90 (1C, C_(α) Cit), 46.69 (1C, CH Fmoc), 38.76 (1C, NCH₂ Cit), 30.56 (1C, C_(β) Val), 28.38, 26.71 (2C, CH₂ Cit), 19.19, 18.24 (2C, CH₃ Val)

1.4. Synthesis of Fmoc-Val-Cit-PAB-OH (4)

Fmoc-Val-Cit-OH (518 mg, 1.04 mmol, 1 eq) and p-aminobenzyl alcohol (256 mg, 2.08 mmol, 2 eq) are dissolved in 18 mL of DCM/MeOH 2/1 and EEDQ (514 mg, 2.08 mmol, 2 eq) is added. The medium is stirred for 15 h in the dark at ambient temperature. The solvents are evaporated, and the yellow solid obtained is triturated with ether. The suspension is sonicated for 5 min and left to stand for 30 min. It is then filtered and the solid is rinsed several times with ether in order to produce a yellow powder (559 mg, 89%).

HRMS: m/z calculated: 601.85499 [M+H]+. found: 602.29758.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 9.97 (s, 1H; NH PAB), 8.10 (d, J=7.0 Hz, 1H; CONH Cit), 7.87-7.18 (m, 13H; Ar Fmoc, Ar PAB, CONH Val), 5.95 (br t, J=5.5 Hz, 1H; NH Cit), 5.40 (s, 2H; NH₂ Cit), 5.09 (t, J=5.5 Hz, 1H; OH PAB), 5.39 (d, J=5.5 Hz, 1H; CH₂ PAB), 4.27-4.19 (m, 4H; CH₂ Fmoc, CH Fmoc, H_(α) Cit), 3.89 (dd, J=8.0 Hz, J=8.0 Hz, 1H; H_(α) Val), 3.03-2.84 (m, 2H; NCH₂ Cit), 1.98-1.92 (m, 1H; H_(β) Val), 1.69-1.27 (m, 4H; CH₂ Cit), 0.84, 0.82 (2d, J=7.0 Hz, 6H; CH₃ Val)

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 171.30, 170.43 (2C, C═O Cit, C═O Val), 158.94 (1C, NH₂CO Cit), 156.17 (1C, C═O Fmoc), 143.94, 143.81, 140.75 (4C, C—Ar Fmoc), 137.55, 137.48 (2C, C—Ar PAB), 127.70, 127.13, 125.40, 120.14 (8C, CH—Ar Fmoc), 126.98, 118.91 (4C, CH—Ar PAB), 65.73 (1C, CH₂ Fmoc), 62.63 (1C, CH₂ PAB), 60.14 (1C, C_(α) Val), 53.11 (1C, C_(α) Cit), 46.72 (1C, CH Fmoc), 38.96 (1C, NCH₂ Cit), 30.48 (1C, C_(β) Val), 29.57, 26.81 (2C, CH₂ Cit), 19.26, 18.31 (2C, CH₃ Val)

1.5. Synthesis of H-Val-Cit-PAB-OH (5)

Fmoc-Val-Cit-PAB-OH (298 mg, 0.495 mmol, 1 eq) is dissolved in 4 mL of DMF and 1 mL of piperidine is added. After reaction for 1 h the medium is evaporated. The viscous solid obtained is triturated with DCM in order to produce a beige solid after filtration (189 mg, 100%).

HRMS: m/z calculated: 601.85499 [M+H]+. found: 602.29758.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 10.04 (s, 1H; NH PAB), 8.16 (d, J=7.5 Hz, 1H; CONH Cit), 7.52, 7.22 (2d, J=8.5 Hz, 4H; Ar PAB), 5.97 (br t, J=5.5 Hz, 1H; NH Cit), 5.40 (s, 2H; NH₂ Cit), 5.10 (br t, J=5.5 Hz, 1H; OH PAB), 4.46-4.40 (m, 3H; CH₂ PAB, H_(α) Cit), 3.52-3.18 (NH₂ Val, water), 3.05-2.87 (m, 3H; H_(α) Val, NCH₂ Cit), 1.95-1.89 (m, 1H; H_(β) Val), 1.71-1.28 (m, 4H; CH₂ Cit), 0.87, 0.77 (2d, J=7.0 Hz, 6H; CH₃ Val)

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 170.48 (2C, C═O Cit, C═O Val), 158.86 (1C, NH₂CO Cit), 137.52, 137.43 (2C, C—Ar PAB), 126.94, 118.95 (4C, CH—Ar PAB), 62.59 (1C, CH₂ PAB), 59.50 (1C, C_(α) Val), 52.53 (1C, C_(α) Cit), 38.08 (1C, NCH₂ Cit), 31.25 (1C, C_(β) Val), 30.10, 26.70 (2C, CH₂ Cit), 19.46, 16.96 (2C, CH₃ Val)

Example 2 Maleimide Derivative as Bioconjugation Head

2.1. Synthesis of 6-maleimidohexanoic acid (6)

Maleic anhydride (1.50 g, 15.25 mmol, 1 eq) and 6-aminocaproic acid (2.00 g, 15.25 mmol, 1 eq) are stirred under argon in 15 mL of acetic acid. The reaction medium is heated under reflux and stirred overnight. The next day, the mixture has turned orange. The acetic acid is coevaporated three times with 75 mL of toluene and a viscous orange-coloured oil is obtained; it is taken up in 150 mL of DCM and then washed with 150 mL of water. The aqueous phase is extracted again (2×150 mL) and the combined organic phases are dried over MgSO₄ and then evaporated under vacuum in order to produce an orange-coloured solid (2.60 g, 81%).

HRMS: m/z calculated: 216.95765 [M+H]⁺. found: 217.09071.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 6.69 (s, 2H; CH═CH), 3.51 (t, J=7.0 Hz, 2H; NCH₂), 2.34 (t, J=7.5 Hz, 2H; CH₂COOH), 1.70-1.55 (m, 4H; NCH₂CH₂CH₂CH₂), 1.38-1.28 (m, 2H; NCH₂CH₂CH₂)

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 179.53 (1C, COOH), 171.00 (2C, C═O MC), 134.21 (2C, CH═CH), 37.71 (1C, NCH₂), 33.86 (1C, CH₂COOH), 28.30 (1C, NCH₂CH₂), 26.24 (1C, NCH₂CH₂CH₂), 24.21 (1C, CH₂CH₂COOH)

2.2. Synthesis of 6-(3,4-dibromomaleimido)hexanoic acid (7)

Maleimidohexanoic acid (200 mg, 0.947 mmol, 1 eq) and sodium acetate (117 mg, 1.421 mmol, 1.5 eq) are put in a tube under argon and 2.9 mL of acetic acid is added. Dibromine (0.07 mL, 1.421 mmol, 1.5 eq) is added at 0° C., the tube is sealed and then heated under reflux for 4 h. The medium is then left to return to ambient temperature and 16 mL of iced water is added. The aqueous phase is extracted with EtOAc (3×20 mL) and the combined organic phases are washed with 40 mL of a solution of sodium thiosulphate, dried over MgSO₄, and coevaporated with toluene. The brown residue obtained is purified by flash chromatography from 0 to 40% of EtOAc in cyclohexane in order to produce a yellow solid (237 mg, 68%).

HRMS: m/z calculated: 368.47706 [M+H]+. found: 367.91255

¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.62 (t, J=7.0 Hz, 2H; NCH₂), 2.36 (t, J=7.5 Hz, 2H; CH₂COOH), 1.72-1.59 (m, 4H; NCH₂CH₂CH₂CH₂), 1.42-1.31 (m, 2H; NCH₂CH₂CH₂)

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 178.18 (1C, COOH), 164.10 (2C, C═O MC), 129.50 (2C, C═C), 39.63 (1C, NCH₂), 33.60 (1C, CH₂COOH), 28.25 (1C, NCH₂CH₂), 26.16 (1C, NCH₂CH₂CH₂), 24.17 (1C, CH₂CH₂COOH)

2.3. Synthesis of 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-OH (8)

6-(3,4-Dibromomaleimido)hexanoic acid (178 mg, 0.482 mmol, 1 eq) and HATU (238 mg, 0.627 mmol, 1.3 eq) are dissolved in 2.2 mL of anhydrous DMF and then 2,6-lutidine (73 μL, 0.627 mmol, 1.3 eq) is added. The medium is stirred for 1 h, then dipeptide (183 mg, 0.482 mmol, 1 eq) is added and the reaction is left overnight with stirring. 1.6 mL of 1M HCl is then added and the aqueous phase is extracted twice with EtOAc. The combined organic phases are washed three times with saturated NaCl solution, and then evaporated. The residue obtained is purified by silica gel column chromatography with a gradient of DCM/MeOH eluent from 15/1 to 9/1 in order to produce a yellow solid (84 mg, 24%).

HRMS: m/z calculated: 729.15941 [M+H]+. found: 729.12412.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 9.91 (s, 1H; NH PAB), 8.08 (d, J=8.0 Hz, 1H; CONH Cit), 7.83 (d, J=8.5 Hz, 1H; CONH Val), 7.54, 7.22 (2d, J=8.5 Hz, 4H; Ar PAB), 5.98 (br t, J=5.5 Hz, 1H; NH Cit), 5.42 (s, 2H; NH₂ Cit), 5.15-5.07 (m, 1H; OH PAB), 4.42-4.33 (m, 3H; CH₂ PAB, H_(α) Cit), 4.19 (dd, J=7.0 Hz, J=8.5 Hz, 1H; H_(α) Val), 3.46-3.27 (NCH₂ MC, water), 3.05-2.89 (m, 2H; NCH₂ Cit), 2.23-2.08 (m, 2H; CH₂CO MC), 2.01-1.91 (m, 1H; H_(β) Val), 1.73-1.16 (m, 10H; CH₂ Cit, NCH₂CH₂CH₂CH₂), 0.85, 0.81 (2d, J=7.0 Hz, 6H; CH₃ Val)

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 172.33, 171.28, 170.39, 163.13 (5C, C═O Cit, C═O Val, C═O MC), 158.92 (1C, NH₂CO Cit), 137.54, 137.42 (2C, C—Ar PAB), 132.30 (2C, C═C), 126.94, 118.86 (4C, CH—Ar PAB), 62.60 (1C, CH₂ PAB), 57.61 (1C, C_(α) Val), 53.08 (1C, C_(α) Cit), 38.96 (1C, NCH₂ MC), 38.68 (1C, NCH₂ Cit), 34.94 (1C, CH₂CO MC), 30.39 (1C, C_(β) Val), 29.39, 27.55, 26.78, 25.70, 24.91 (5C, NCH₂CH₂CH₂CH₂, CH₂ Cit), 19.26, 18.18 (2C, CH₃ Val)

2.4. Synthesis of 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-OCOOPNP (9)

6-(3,4-Dibromomaleimido)hexanamide-Val-Cit-PAB-OH (42 mg, 57.5 μmol, 1 eq) is dissolved in 0.85 mL of anhydrous pyridine. PNPOCOCl (35 mg, 172.5 μmol, 3 eq) is dissolved in 0.6 mL of anhydrous DCM and then added to the solution of 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-OH in pyridine at 0° C. After 1 h there is no longer any of the starting product. The pyridine is coevaporated three times with toluene and then the residue is purified by column chromatography with a gradient of DCM/MeOH eluent from 15/1 to 8/2 in order to produce an orange-coloured solid (5.9 mg, 11%).

The product is used directly for grafting on the molecule of interest (i.e. drug, fluorochrome, etc.).

2.5. Synthesis of 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-MMAE (9-MMAE)

6-(3,4-Dibromomaleimido)hexanamide-Val-Cit-PAB-OCOOPNP (9.0 mg, 10 μmol, 1 eq) is dissolved under argon at 25° C. in 1 mL of anhydrous DMF/pyridine mixture: 4/1. DIPEA (1.4 μL, 1.2 eq), HOBt (1.83 mg, 1 eq) and MMAE (7.2 mg, 10 μmol, 1 eq) are added to this solution successively. The mixture is stirred for 24 h (monitoring with HPLC) and the crude reaction product is purified by semipreparative HPLC in order to provide the linker 9-MMAE (4.3 mg, 29%) in the form of yellow solid, the conformity of which is verified by mass spectroscopy.

Example 3 Benzene Derivative as Bioconjugation Head

3.1. Methyl 3,5-bis(bromomethyl)benzoate (10)

Methyl 3,5-bis(bromomethyl)benzoate (1.00 g, 6.09 mmol, 1 eq) is dissolved in anhydrous CCl₄ (25 mL) and the mixture is heated under reflux. NBS (2.38 g, 13.40 mmol, 2.2 eq) is added in five equal portions over 8 h followed by the addition of a few milligrams of Bz₂O₂. The mixture is stirred overnight and then filtered. The filtrate is washed with water (50 mL), saturated NaHCO₃ solution (25 mL) and brine (30 mL). The organic layer is dried over MgSO₄ and evaporated. The residue is dissolved in 25 ml of anhydrous THF and diethyl phosphite (1.7 mL, 13.40 mmol, 2.2 eq) and anhydrous DIPEA (2.3 mL, 13.40 mmol, 2.2 eq) is added at 0° C. The mixture is then stirred at ambient temperature for two days. It is evaporated and 50 mL of iced water is added and extracted with ether (3×25 mL). The combined organic layers are washed with 1M HCl solution (25 mL), brine (25 mL) and are dried over MgSO₄. Evaporation of the solvent under reduced pressure produced a residue, which is purified by flash chromatography in a gradient of cyclohexane at 5% of EtOAc in cyclohexane in order to produce 10 in the form of a white solid (0.83 g, 42%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) 8.00 (d, J=2.0 Hz, 2H; H-2, H-6), 7.62 (t, J=2.0 Hz, 1H; H-4), 4.50 (s, 4H; CH₂Br), 3.94 (s, 3H; COCH₃).

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 166.07 (1C, COO), 139.09 (2C, C-3, C-5), 134.00 (1C, CH-4), 131.55 (1C, C-1), 130.18 (2C, CH-2, CH-6), 52.57 (1C, OCH₃), 31.99 (2C, CH₂Br).

3.2. General Procedure 1 for Saponification with Lithium Hydroxide

The ester (1 mmol) is dissolved in THF (20 mL), and LiOH is added (2.5 eq). The reaction mixture is stirred at ambient temperature until the end of the reaction (generally 3 h, verified by monitoring with TLC). Then 1M of HCl is added until the pH of the solution becomes acid (pH=2), and the mixture is extracted with EtOAc (3×25 mL). The combined organic layers are dried over MgSO₄. Evaporation of the solvent under reduced pressure makes it possible to obtain the corresponding acid.

3.3. General Procedure 2 for Peptide Coupling Using HATU

The acid (0.2 mmol) is dissolved in anhydrous DMF (1 mL) under argon, to which HATU (1.2 eq) and 2,6-lutidine (1.3 eq) are added at ambient temperature. The reaction mixture is stirred for 15 to 40 min at ambient temperature, then a solution of amine (1.0 eq) in DMF (1 mL) is added, and the mixture is stirred until the end of the reaction (typically 4 hours) at ambient temperature under argon. Then the mixture is diluted by adding a solution of 15% citric acid and EtOAc, and then filtered. The filtrate is extracted three times with EtOac, washed with saturated solutions of NaCl and NaHCO₃. The organic layer is dried (MgSO₄) and evaporated under reduced pressure. The crude product is purified by flash chromatography in order to produce the desired amide.

3.4. General Procedure 3 for Bromination Using Phosphorus Tribromide

The alcohol is dissolved in anhydrous ACN under argon, then PBr3 is added and the reaction mixture is heated at 60° C. under argon for 16 h. After cooling, the reaction mixture is diluted with water, and extracted three times with EtOAc. The combined organic layers are washed with brine and dried over MgSO₄. Evaporation of the solvent under reduced pressure produced the desired corresponding brominated compound.

3.5. 3-(Bromomethyl)-5-(hydroxymethyl)benzoic acid (11)

Compound 11 is prepared from 10 (317 mg, 0.98 mmol) by general procedure 1 in order to produce 11 (160 mg, 66%) in the form of a white solid.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 8.21-7.97 (m, 2H, H_(arom)), 7.69 (d, J=2.0 Hz, 1H, H_(arom)), 4.64 (d, J=3.0 Hz, 2H, CH₂OH), 4.53 (d, J=3.0 Hz, 2H, CH₂Br).

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 170.10 (1C, COO), 139.34 (1C, C-5), 137.86 (1C, C-3), 134.91 (1C, CH-4), 134.45 (1C, C-1), 130.71 (1C, C-2), 130.28 (1C, C-3), 45.10 (1C, CH₂OH), 31.81 (1C, CH₂Br).

3.6. Methyl 6-(3-(bromomethyl)-5-(hydroxymethyl)benzamido)hexanoate (12)

Compound 12 is prepared from 11 (50 mg, 0.20 mmol) by general procedure 2 in order to produce 12 (41 mg, 54%) in the form of a white solid.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.78-7.50 (m, 3H, H_(arom)), 6.35 (s, 1H, NH), 4.60 (d, J=3.1 Hz, 2H, CH₂OH), 4.49 (d, J=3.1 Hz, 2H, CH₂Br), 3.66 (s, 3H, CH₃), 3.47 (dd, J=13.0, 6.9 Hz, 2H, CH₂-5), 2.34 (t, J=7.3 Hz, 2H, CH₂-1), 1.78-1.54 (m, 4H, CH₂-2, CH₂-3), 1.52-1.33 (m, 2H, CH₂-4).

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 174.28 (1C, COO), 166.53 (1C, CONH), 139.08 (1C, C_(Ar)-5), 138.75 (1C, C_(Ar)-3), 136.08 (1C, C_(Ar)-1), 131.98 (1C, C_(Ar)-2), 127.62 (1C, C_(Ar)-2), 127.17 (1C, C_(Ar)-6), 51.71 (1C, CH₂OH), 45.36 (1C, CH-5), 39.90 (1C, CH-1), 33.88 (1C, CH₂Br), 32.19 (1C, CH₃), 29.21 (1C, CH-2), 26.40 (1C, CH-3), 24.37 (1C, CH-4).

3.7. 6-(3-(Bromomethyl)-5-(hydroxymethyl)benzamido)hexanoic acid (13)

Compound 13 is prepared from 12 (215 mg, 0.58 mmol) by general procedure 1 in order to produce 13 (160 mg, 77%) in the form of an electrostatic white solid.

HRMS: m/z calculated: 356.0576 [M+H]+. found: 356.0578.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 12.0 (bs, 1H, COOH), 8.57 (t, J=5.5 Hz, 1H, NH), 7.92-7.61 (m, 3H, H_(arom)), 4.90-4.67 (m, 4H, CH₂Br, CH₂OH), 3.24 (dd, J=12.8, 6.8 Hz, 2H, CH₂-5), 2.21 (t, J=7.3 Hz, 2H, CH₂-1), 1.62-1.42 (m, 4H, CH₂-2, CH₂-3), 1.40-1.23 (m, 2H, CH₂-4).

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 174.50 (1C, COO), 165.21 (1C, CONH), 138.35 (1C, C_(Ar)-5), 135.60 (1C, C_(Ar)-3), 133.92 (1C, C_(Ar)-1), 131.59 (1C, C_(Ar)-2), 127.58 (1C, C_(Ar)-6), 45.48 (1C, CH₂OH), 39.15 (1C, CH-5), 33.62 (2C, CH-1, CH₂Br), 28.81 (1C, CH-2), 26.05 (1C, CH-3), 24.27 (1C, CH-4).

3.8. 6-(3,5-bis(Bromomethyl)benzamido)hexanoic acid (14)

Compound 14 is prepared from 13 (20 mg, 0.006 mmol) by general procedure 3 in order to produce 14 (24 mg, quantitative) in the form of an off-white solid.

HRMS: m/z calculated: 419.9732 [M+H]+. found: 419.9808.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 11.8 (bs, 1H, COOH), 8.57 (s, 1H, NH), 7.92-7.62 (m, 3H, H_(arom)), 4.78 (dd, J=20.8, 3.3 Hz, 4H, 2×CH₂Br), 3.32-3.06 (m, 2H+water, CH₂-5), 2.21 (t, J=7.3 Hz, 2H, CH₂-1), 1.65-1.41 (m, 4H, CH₂-2, CH₂-3), 1.40-1.23 (m, 2H, CH₂-4).

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 174.50 (1C, COO), 165.21 (1C, CONH), 138.37 (1C, C_(Ar)-5), 135.58 (1C, C_(Ar)-3), 132.00 (1C, C_(Ar)-1), 131.58 (1C, C_(Ar)-2), 127.58 (1C, C_(Ar)-6), 45.57 (1C, CH₂OH), 39.08 (1C, CH-5), 33.73 (3C, CH-1, CH₂Br), 28.89 (1C, CH-2), 26.04 (1C, CH-3), 24.32 (1C, CH-4).

3.9. 6-(3,5-bis(Bromomethyl)benzamido)hexanamide-Val-Cit-PAB-OH (15)

Compound 15 is prepared from 14 (124 mg, 0.29 mmol) by general procedure 2 in order to produce 15 (30 mg, 13%) in the form of a beige solid.

HRMS: m/z calculated: 781.1846 [M+H]+. found: 781.1907.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 9.95 (s, 1H, NH PAB), 8.71-8.50 (m, 1H, CONH Cit), 8.10 (d, J=7.5 Hz, 1H, CONH Val), 8.04-7.55 (m, 3H, H_(arom)), 7.55, 7.22 (2d, J=8.5 Hz, 4H, Ar PAB), 6.06 (t, J=5.6 Hz, 1H, NH Cit), 5.45 (br s, 2H, NH₂ Cit), 5.12 (br s, 1H, OH PAB), 4.82 (s, 4H, 2×CH₂Br), 4.46-4.32 (m, 3H, CH₂ PAB, H_(α) Cit), 4.26-4.13 (m, 1H, H_(α) Val), 3.24 (dd, J=12.8, 6.6 Hz, CH₂-5), 3.10-2.84 (m, 2H, NCH₂ Cit), 2.29-2.07 (m, 2H, CH₂-1), 1.97 (dq, J=13.7, 6.6 Hz, 1H, Hp Val), 1.80-1.05 (m, 10H, CH₂CH₂ Cit, NCH₂CH₂CH₂CH₂), 0.83 (dd, J=8.9, 7.0 Hz, 6H, CH₃ Val).

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 172.44, 171.29, 170.42, 165.18, 165.02, 158.94, 151.89, 151.12, 139.24, 138.32, 138.28, 137.56, 137.41, 135.59, 135.45, 134.46, 133.79, 132.58, 131.55, 129.41, 128.86, 128.73, 128.60, 127.59, 126.92, 121.31, 120.73, 118.86, 81.78, 62.60, 57.67, 53.08, 48.61, 45.49, 45.36, 38.56, 35.17, 30.37, 29.36, 28.86, 26.79, 26.19, 25.23, 19.26, 18.23.

Example 4 Pyridine Derivative as Bioconjugation Head

4.1. Methyl 2,6-bis(hydroxymethyl)isonicotinate (16)

Methylisonicotinate (2.12 mL, 18 mmol) is dissolved under argon in a mixture of MeOH/H₂O: 1/1 (40 mL), then H₂SO₄ (78 μl) is added and the mixture is stirred for 30 min. Then ammonium persulphate (32 g, 144 mmol) and iron chloride tetrahydrate (0.89 g, 4.5 mmol) are added carefully at 0° C. and the mixture is heated at 50° C. overnight. After the reaction mixture has cooled, the suspension is filtered and the solid is washed with a small quantity of EtOAc. The filtrate is concentrated under reduced pressure until all the MeOH has been removed. The medium is made basic with Na₂CO₃ and is then extracted with EtOAc. The organic layer is dried over MgSO₄, filtered and concentrated under reduced pressure. The crude product is purified by flash chromatography in a DCM gradient of 10% of MeOH in DCM in order to produce 16 in the form of a dark brown solid (0.54 g, 15%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.82 (s, 2H, H_(arom)), 4.88 (s, 4H, CH₂OH), 3.98 (s, 3H, CH₃).

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 165.57 (1C, COOCH₃), 160.00 (1C, C-1), 150.47 (2C, C-3, C-5), 118.77 (2C, C-2, C-6), 64.54 (2C, CH₂OH), 52.98 (1C, CH₃).

4.2. Methyl 2,6-bis(bromomethyl)isonicotinate (17)

Compound 17 is prepared from 16 (120 mg, 061 mmol) by general procedure 3 in order to produce 17 (141 mg, 72%) in the form of a light pink solid.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.92 (s, 2H, H_(arom)), 4.58 (s, 4H, CH₂Br), 3.98 (s, 3H, CH₃).

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 164.8 (1C, COOCH₃), 158.0 (1C, C-1), 139.8 (2C, C-3, C-5), 122.3 (2C, C-2, C-6), 53.0 (1C, CH₃), 32.8 (2C, CH₂Br).

4.3. 2,6-bis(Bromomethyl)isonicotinic acid (18)

Compound 18 is prepared from 17 (137 mg, 0.42 mmol) by general procedure 1 in order to produce 18 (133 mg, quantitative) in the form of an orange-coloured solid.

HRMS: m/z calculated: 307.88441 [M+H]+. found: 307.8917.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 7.92 (s, 2H, H_(arom)), 4.79 (s, 4H, CH₂Br), 3.35 (s, 3H, CH₃).

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 165.9 (1C, COOCH₃), 158.6 (1C, C-1), 141.0 (2C, C-3, C-5), 122.7 (2C, C-2, C-6), 34.3 (2C, CH₂Br).

4.4. Methyl 6-(2,6-bis(bromomethyl)isonicotinamido)hexanoate (19)

Compound 19 is prepared from 18 (260 mg, 0.84 mmol) by general procedure 2 in order to produce 19 (218 mg, 59%) in the form of a yellow solid.

HRMS: m/z calculated: 434.9841 [M+H]⁺. found: 434.9906.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.70 (s, 2H, H_(arom)), 6.41 (s, 1H, NH), 4.58 (s, 4H, CH₂Br), 3.69 (s, 3H, OCH₃), 3.50 (dd, J=13.5, 7.0 Hz, 2H, CH₂-5), 2.36 (t, J=7.0 Hz, 2H, CH₂-1), 1.68 (dq, J=13.5, 7.0 Hz, 4H, CH₂-2, CH₂-4), 1.54-1.34 (m, 2H, CH₂-3).

¹³C NMR (75 MHz, CDCl₃): δ (ppm) 173.1 (1C, COOCH₃), 157.8 (1C, CONH), 155.0 (2C_(pyridine), C-4, C-6), 120.3 (2C_(pyridine), C-3, C-5), 51.7 (1C, CH₃), 39.8 (1C, CH₂-5), 33.6 (1C, CH₂-1), 32.9 (2C, CH₂Br), 28.8 (1C, CH₂-4), 26.1 (1C, CH₂-3), 24.0 (1C, CH₂-2).

4.5. 6-(2,6-bis(Bromomethyl)isonicotinamido)hexanoic acid (20)

Compound 20 is prepared from 19 (216 mg, 0.50 mmol) by general procedure 1 in order to produce 20 (201 mg, 96%) in the form of an off-white solid.

HRMS: m/z calculated: 420.9684 [M+H]⁺. found: 420.9748.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 12.01 (s, 1H, COOH), 8.83 (t, J=6.2 Hz, 1H, NH), 7.84 (s, 2H, H_(arom)), 4.74 (s, 4H, CH₂Br), 3.6 (dd, J=12.6, 6.2 Hz, 2H, CH₂-5), 2.21 (t, J=7.3 Hz, 2H, CH₂-1), 1.67-1.43 (m, 4H, CH₂-2, CH₂-4), 1.54-1.34 (dd, J=8.2, 4.6 Hz, 2H, CH₂-3).

¹³C NMR (75 MHz, DMSO-d₆): δ (ppm) 174.9 (1C, COOH), 164.3 (1C, CONH), 158.0 (2C_(pyridine), C—CH₂Br), 144.6 (1C_(pyridine), C—CONH), 121.3 (2C_(pyridine), CH), 39.5 (1C, CH₂-5), 34.6 (2C, CH₂Br), 34.0 (1C, CH₂-1), 29.0 (1C, CH₂-4), 26.4 (1C, CH₂-3), 24.7 (1C, CH₂-2).

4.6. 4-Nitrophenyl 6-(2,6-bis(bromomethyl)isonicotinamido)hexanoate (21)

Under argon at ambient temperature, the acid 20 (70 mg, 0.17 mmol) is dissolved in anhydrous CH₂Cl₂ (2.1 mL), and DMAP in catalytic quantities, p-nitrophenol (30 mg, 1.3 eq) and DCC (45 mg, 1.3 eq) are added successively to this mixture. The reaction mixture is stirred for 16 hours at ambient temperature under an argon atmosphere, and then filtered. The filtrate is concentrated under reduced pressure and the crude product is purified by flash chromatography in a gradient of cyclohexane at 40% of EtOAc in cyclohexane in order to produce 21 in the form of a pale yellow solid (56 mg, 62%). The product is verified by ¹H NMR and is used directly for the next step.

HRMS: m/z calculated: 541.9848 [M+M]⁺. found: 541.9918.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 8.34-8.21 (m, 2H, H_(arom PNP)), 7.67 (s, 2H, H_(arom pyridine)), 7.35-7.18 (m, 2H, H_(arom PNP)), 6.29 (s, 1H, NH), 4.56 (s, 4H, CH₂Br), 3.52 (dd, J=13.0, 7.0 Hz, 1H, CH₂-5), 2.66 (t, J=7.0 Hz, 1H, CH₂-1), 1.91-1.45 (m, 6H, CH₂-2, CH₂-3, CH₂-4).

4.7. 6-(2,6-bis(Bromomethyl)isonicotinamido)hexanamide-Val-Cit-PAB-OH (22)

4-Nitrophenyl 6-(2,6-bis(bromomethyl)isonicotinamido)hexanoate 21 (56 mg, 0.10 mmol) is dissolved in 2.0 mL of DMF under an argon atmosphere at ambient temperature. After 5 min, H-Val-Cit-PAB-OH (20 mg, 0.05 mmol) is added, and the reaction mixture is stirred for 5 h. The DMF is then removed by lyophilization, and the crude product is purified by flash chromatography in a DCM gradient at 10% of MeOH in DCM in order to produce 22 in the form of a pale yellow solid (8.5 mg, 20%). The product is verified by ¹H NMR and is used directly for the next step.

HRMS: m/z calculated: 781.1798 [M+M]⁺. found: 782.1873.

¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 9.90 (s, 1H, NH PAB), 8.83 (d, J=5.7 Hz, 1H, CONH Cit), 8.07 (d, J=7.5 Hz, 1H, CONH Val), 7.84 (s, 4H, 2CH_(pyridine)), 7.54, 7.22 (2d, J=8.5 Hz, 4H, Ar PAB), 5.98 (br t, J=5.7 Hz, 1H, NH Cit), 5.42 (br s, 2H, NH₂ Cit), 5.11 (br s, 1H, OH PAB), 4.73 (s, 4H, 2×CH₂Br), 4.48-4.31 (m, 3H, CH₂ PAB, H_(α) Cit), 4.19 (dd, J=7.0 Hz, J=8.5 Hz, 1H, H_(α) Val), 3.48-3.12 (water, CH₂-5), 3.06-2.89 (m, 2H, NCH₂ Cit), 2.18 (dd, J=14.5, 6.5 Hz, 2H, CH₂-1), 2.04-1.86 (m, 1H, H_(β) Val), 1.79-1.21 (m, 10H, CH₂CH₂ Cit, NCH₂CH₂CH₂CH₂), 0.83 (dd, J=8.9, 6.8 Hz, 6H, CH₃ Val).

4.8. Methyl hydrochloride 6 aminohexanoate (23)

Under an argon atmosphere at 0° C., SOCl₂ (1.22 mL, 16.7 mmol) is added slowly to MeOH (10 ml) and the reaction mixture is stirred for 20 min. 6-Aminocaproic acid (1.00 g, 7.6 mmol) is added to this solution at 0° C., and the mixture is stirred for 3.5 h. The volatiles are removed under reduced pressure, and the residue is recrystallized from heptane/EtOAc/MeOH mixture in order to obtain the desired ester 23 (1.36 g) in a quantitative yield.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.84 (s, 1H, NH), 3.58 (s, 3H, OCH₃), 2.85-2.65 (m, 2H, CH₂-1), 2.30 (t, J=7.3 Hz, 2H, CH₂-1), 1.65-1.36 (m, 4H, CH₂-2, CH₂-4), 1.31 (ddd, J=11.9, 7.3, 2.3 Hz, 2H, CH₂-3).

Example 5 Bioconjugation—Reconstruction of the Disulphide Bridges 5.1. General Procedure 4 for Bioconjugation

A solution of the desired antibody is taken. The interchain disulphide bridges are reduced by TCEP (verified by RP-HPLC), then a solution of the appropriate linker is added. After a suitable reaction time (disappearance of the light and heavy chains and reconstruction of the disulphide bridges are monitored by RP-HPLC), the modified crude antibody is purified on columns of Sephadex PD-10.

5.2. TRASTUZUMAB-(maldiBr-linker7)₄ 24

Trastuzumab is reduced and then modified according to general procedure 4, using the noncleavable linker 7, 3,4-dibromomaleimide at 37° C. for 1 h:

A solution of TCEP (20 eq) in PBS buffer is added to a solution of trastuzumab. A fraction is taken for analysis, to confirm rupture of the disulphide bridges. The mixture is stirred for 15 minutes, then a solution of 6-(3,4-dibromomaleimido)hexanoic acid (20 eq) in DMSO is added. The reaction mixture is stirred for 30 minutes, and then analyzed by RP-HPLC (FIG. 5).

MALDI-TOF mass analysis (FIG. 2) confirms that the reaction mixture (green) contains at 89%, the species resulting from grafting of 4 linkers 6-(3,4-dibromomaleimido)hexanoic acid on the antibody; the grafting average is 4.0.

5.3. TRASTUZUMAB-(maldiBr-linker8)₄ 25

Trastuzumab is reduced and then modified according to general procedure 4, using the cleavable linker 8, 3,4-dibromomaleimide at 35° C. for 1 h:

A solution of TCEP (20 eq) in PBS buffer is added to a solution of trastuzumab. The mixture is stirred for 15 minutes, and then a solution of 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-OH (20 eq) in DMSO is added. The reaction mixture is stirred for 30 minutes, and then, after purification on a PD-10 column to remove the chemical reagents, the mixture is analyzed by RP-HPLC (FIG. 6), and by HIC-HPLC (FIG. 3).

5.4. TRASTUZUMAB-(maldiSPh-linker26)₄ 27

Linker 26 is synthesized in a manner similar to that of WO2013132268, for purposes of comparison between reconstruction of the disulphide bridges using linker 8 dibromomaleimide or linker 26 dithiophenylmaleimide.

Trastuzumab is reduced and then modified according to general procedure 4, using the cleavable linker 8, 3,4-dithiophenylmaleimide at 41° C. for 48 h:

The reaction mixture is then analyzed by RP-HPLC (FIG. 7).

5.5. TRASTUZUMAB-(PydiMediBr-linker20)₄ 28

Trastuzumab is reduced and then modified according to general procedure 4, using the non-cleavable linker 20, 6-(2,6-bis(bromomethyl)isonicotinamido)hexanoic acid at 41° C. for 24 h:

The reaction mixture is then analyzed by RP-HPLC (FIG. 8).

5.6. TRASTUZUMAB-(PydiMediBr-linker22)₄ 29

Trastuzumab is reduced and then modified according to general procedure 4, using the cleavable linker 22, 6-(2,6-bis(bromomethyl)isonicotinamido)hexanamide-Val-Cit-PAB-OH at 41° C. for 24 h:

The reaction mixture is then analyzed by RP-HPLC (FIG. 9).

5.7. TRASTUZUMAB-(PhdiMediBr-linker15)₄ 30

Trastuzumab is reduced and then modified according to general procedure 4, using the cleavable linker 15, 6-(3,5-bis(bromomethyl)benzamido)hexanamide-Val-Cit-PAB-OH at 41° C. for 72 h:

The reaction mixture is then analyzed by RP-HPLC (FIG. 10).

5.8. RITUXIMAB-(maldiBr-linker7)₄ 31

Rituximab is reduced and then modified according to general procedure 4, using the non-cleavable linker 7 at 37° C. for 1 h:

The reaction mixture is then analyzed by RP-HPLC (FIG. 11).

5.9. TRASTUZUMAB-(maldiBr-linker9-MMAE)₄ 32

Trastuzumab is reduced and then modified according to general procedure 4, using the cleavable linker 9-MMAE, 6-(3,4-dibromomaleimido)hexanamide-Val-Cit-PAB-MMAE at 37° C. for 24 h:

The reaction mixture is then analyzed by RP-HPLC (FIG. 12).

5.10. Additional Experimental Data for Analytical Data

a—RP-HPLC

The ADCs were analyzed under denaturing and reducing conditions, with an AERIS WIDEPORE 3.6 μM XB-C8 column (250×4.6 mm). The flow rate was 1 mL/min, furnace temperature of 80° C., with a quantity injected of 20 μL per sample. Regarding the method of elution: with two solvents: A was 0.05% trifluoroacetic acid in water and B: 0.04% trifluoroacetic acid in acetonitrile. The method was as follows: 25% B isocratic for 3 min, a linear gradient of 25 min from 25 to 50% B, a linear gradient of 2 min from 50 to 95% B, a linear gradient of 1 min from 95 to 25% B, then 25% B isocratic for 8 min.

b—HIC-HPLC

The ADCs were analyzed under non-denaturing conditions, with a TOSOH BIOSCIENCE 2.5 μM T SKgel butyl-NPR column (100×4.6 mm). The flow rate was 1 mL/min, furnace temperature of 80° C., with an injected quantity of 30 μL per sample. Regarding the method of elution with two solvents: A: 1.5 M (NH₄)₂SO₄ in 50 mM PBS phosphate buffer (pH 7) and B: 50 mM PBS phosphate buffer/iProH mixture: 80/20. The method was as follows: a linear gradient of 44 min from 0% B to 80% B, then a linear gradient of 1 min from 80 to 0% B, then 0% B isocratic for 10 min. 

1-25. (canceled)
 26. A product that corresponds to formula I selected from formulae IB, IB1, IB2 and IA:

where in formulae IA and IB: X is Br or I, or a

 group, or a

 group, X_(a) being a halogen or a nucleofuge. A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

 group, a

 group or a

 group X₁ is a C═O or an NH group or a single bond X₂ is an NH group or a C═O or a single bond X₃ is an oxygen or a single bond s is equal to 1, 2 or 3 r is equal to 0, 1 or 2 it being understood that the sum r+s is equal to 3 n₁ is an integer equal to 0 or 1; n₂ is an integer equal to 1, 2 or 3 and n₃ is an integer equal to 1, 2 or 3 n₄ is an integer equal to 1, 2, 3 or 4 W represents an -L radical which represents a linker body comprising a terminal reactive function, an -FL fluorophore group used in diagnostics or analysis, involving detection of fluorescence preferably selected from rhodamine or a derivative thereof, preferably rhodamine B, fluorescein isothiocyanate (FITC), a Cy dye preferably selected from Cy5, Cy5.5, Cy7, an Alexa fluor dye preferably selected from Alexa Fluor 647, 700 or 750, Texas Red, Nile Blue A, allophycocyanin (APC) and conjugates thereof with other fluorochromes in particular those mentioned above, phycoerythrin (PE) and conjugates thereof with other fluorochromes in particular those mentioned above, an R* radioactive radical which preferably contains either a bifunctional ligand, optionally bimodal, preferably selected from the derivatives of DOTA, DTPA, C-DOTA, NODAGA, NETA, C-NETA, DEPA, C-DEPA, TETA, TE2A, HYNIC, DAT, MAMA, or a bifunctional ligand preferably selected from one of the acyclic chelating agents from the list constituted by EDTA, CyEDTA, EDTMP, DTPMP, DTPA, CyDTPA, Cy₂DTPA, DTPA-MA, DTPA-BA, BOPA, or a bifunctional ligand preferably selected from one of the macrocyclic chelating agents from the list constituted by DOTA, TRITA, TETA, DOTA-MA, DO3A-HP, DOTMA, DOTA-pNB, DOTP, DOTMP, DOTEP, DOTMPE, F-DOTPME, DOTPP, DOTBzP, DOTA-monoamide, p-NCS-DOTA, p-NCS-PADOTA, BAT, DO3TMP-Monoamide, p-NCS-TRITA, NOTA, CHX-A″-DTPA, or a bifunctional chelating agent of the cationic, anionic, neutral or cleavable type, or a radionuclide preferably selected from ⁶⁷Cu, ⁶⁴Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁷Au, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁰⁵Rh, ¹⁶⁵Ho, ¹⁶⁶Ho, ¹⁶¹Tb, ¹⁴⁹Pm, ⁴⁴Sc, ⁴⁷Sc, ⁷⁰As, ⁷¹As, ⁷²As, ⁷³As, ⁷⁴As, ⁷⁶As, ⁷⁷As, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, ^(117m)Sn, ⁶⁷Ga, ²⁰¹Tl, ¹²³I, ¹³¹I, ¹⁶⁰Gd, ¹⁴⁸Nd, ⁸⁹Sr, ²¹¹At, a cytotoxic drug M selected from a chemotherapeutic agent or a toxin, in particular monomethyl auristatin E or monomethyl auristatin F, an -L-M radical in which L and M have the meanings given above, P is a protein comprising at least one disulphide bridge and t represents an integer from 1 to 15, preferably from 1 to 6 and 13 as well as the derivatives of the carboxylic acid function or functions such as the salts, esters or amides, preferably the amide formed with butanimide.
 27. Product according to claim 26, wherein the

group is selected from the following radicals:

and wherein the

group is selected from the following radicals:

radicals in which X is Br or I, in the triad of atoms (T,Y,Z) each of these atoms represents, independently of the other two atoms, a carbon or nitrogen atom, in particular T is a nitrogen atom and Y and Z represent CH.
 28. Products according to claim 26 corresponding to general formula IA1:

or IB1:

in which X, A, X1, X2, X3, n₁, n₂, n₃, r, s, t, L and P have the meanings stated in claim
 26. 29. Product according to claim 26, in which the linker body L corresponds to general formula III, IIIa or IIIb:

in which K represents either a

 radical representing a sequence of n₅ amino acids, which can be identical or different, natural or non-natural, or a cleavage site recognized by an enzyme, or a hydrazino radical optionally coupled to the

 radical or K represents a saccharide group preferably selected from a beta-glucuronic acid, a beta-D-galactose, a beta-D-glucose, an alpha-D-mannose, an N-acetyl-D-glucosaminyl, an N-acetyl-D-galactosaminyl, a D-glucuronyl, an L-iduronyl, a D-glucopyranosyl, a D-galactopyranosyl, a D-mannopyranosyl or L-fucopyranosyl. X represents a hydrogen or an NO₂ group, A preferably represents a hydrogen atom, Y represents a

 chain or a single bond or a chemical spacer preferably selected from the group formed by the linear or branched alkyl radical having from 1 to 30 carbon atoms optionally interrupted by one or more oxygen, sulphur or nitrogen atoms, for example a polyethylene glycol. Z represents a leaving group, preferably selected from the group comprising para nitro or para cyano phenyloxy, in particular one of the following radicals:

or a chlorine atom and n₅ represents an integer from 1 to
 6. 30. Product according to claim 29 in which the

radical is such that n₅ is equal to 1, 2, 3, 4 or 5, preferably 2 or 3 and AA preferably represents a valine and a citrulline; or a phenylalanine and a lysine; or a valine and an aspartic acid; or a lysine and a methionine; or a lysine and an asparagine; or a proline and an isoleucine; or a proline and a lysine; or a lysine and a methionine; or a lysine and an asparagine; or a valine and a lysine; or an alanine and a lysine; or a phenylalanine and a lysine; or two phenylalanines and a lysine; or an alanine, a phenylalanine and a lysine; two arginines; a lysine and an arginine; a glutamic acid, a glycine and an arginine; two glycines and an arginine.
 31. Product according to claim 29 in which K represents a site of cleavage by an enzyme selected from the enzymes of the cathepsin B, cathepsin C, cathepsin D type, the enzymes selected from plasmin, lysosomal protease, lysosomal enzyme, or urokinase plasminogen activator (uPA), elastase, proteinase 3, cathepsin G; or K represents a recognition site of cleavage by an enzyme of the matrix metalloproteinase (MMP) type preferably selected from collagenase 1, 2 and 3, gelatinase a and b, stromelysin 1 and 2, matrilysin 1, 2 and 3, macrophage metalloelastase, membrane MMP, enamelysin, CA-MMP, epilysin and PSMA. or K represents a recognition site of cleavage by an esterase, a carboxylesterase, alkaline phosphatases, proteases, peptidases, cathepsins, glucosidase, galactosidase, beta-D-galactosidase, induronidase, beta-glucuronidase, mannosidase, N-acetyl-D-glucosaminidase or N-acetyl-D-galactosaminidase.
 32. Products according to claim 26 corresponding to general formula IA2:

or IB2:

in which X, A, X1, X2, X3, n₁, n₂, n₃, r, s, t, L, P and M have the meanings stated in claim
 26. 33. Product according to claim 26, in which the drug M is selected from the group constituted by duocarmycin and its analogues, dolastatins, combretastatin, calicheamicin, N-acetyl-□-calicheamycin (CMC), a calicheamycin derivative, maytansine and analogues thereof, DM-I, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin, disorazole, the epothilones, Paclitaxel, docetaxel, Topotecan, echinomycin, estramustine, cemadotine, eleutherobin, methopterin, actinomycin, daunorubicin, the daunorubicin conjugates, mitomycin C, mitomycin A, vincristine, retinoic acid, camptothecin, a camptothecin derivative, SN38, maytansine, a derivative of the maytansinoid type, DM1, DM4, TK1, amanitin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, methotrexate, ilomedine, aspirin, an IMIDs, lenalidomide, pomalidomide.
 34. Product according to claim 26, in which the toxin is selected from pseudomonas exotoxin (PE), deBouganin, Bouganin, diphtheria toxin (DT) and ricin.
 35. Product corresponding to formula (IB2).

in which X, A, X1, X2, X3, n₁, n₂, n₃, r, s, t, L, P and M have the meanings stated in claim
 26. 36. Product according to claim 26 in which the protein P comprising at least one disulphide bridge, can be an agonist or an antagonist, an antibody or an antibody fragment, a fusion protein comprising an antibody or an antibody fragment, an albumin, or a peptide.
 37. Product according to claim 36 in which the antibody is a chimeric monoclonal antibody, humanized or human, monospecific or bispecific.
 38. Product according to claim 36, in which the antibody fragment is selected from the group constituted by: Fab, F(ab)′2, Fc, F′c, pFc′, ScFv, Fv, Fd, Fabc, diabody, minibody, ScFv-Fc or ScFv-Fv.
 39. Product according to claim 36, in which the antibody or antibody fragment is directed against a tumour antigen or inflammation antigen.
 40. Product according to claim 36, in which the antibody or antibody fragment is directed either against one of the antigens of the cluster of differentiation (CD), the identification number of which varies between CD1a and CD363 and is preferably selected from CD3, CD4, CD13, CD19, CD20, CD21, CD22, CD25, CD30, CD31, CD33, CD34, CD37, CD39, CD40, CD44, CD47, CD52, CD56, CD66e, CD70, CD72, CD73, CD74, CD79, CD79b, CD80, CD86, CD117, CD138, CD194, CD205, CD227 or CD248 or the antibody or antibody fragment is directed against one of the antigens selected from the list formed by CA125, G250, GD2, HLA-DR□, MUC1, VEGF, VEGFA, VEGF-R1/2, TRAIL-R2 (DR5), EpCAM, GPIIb, GPIIIa, TNF alpha, TNFR, TNT, Lewis Y, EGFR, HER-2, HER-3, HER-3 MM-111, HER-4, homodimer or heterodimer between members of the erbb_(n) family, n being an integer comprised between 1 and 4, AXL, Protein F, IgE-Fc, VEGF-A, integrin 4, integrin alpha4beta7, integrin alphaV, C5, IL-6R, IL-6Ralpha, IL12, IL15, IL18, IL23, IL-1beta, IL-1, TPO-R, GPNMB, PSMA, PSA, PAP, PSM, integrin alpha_(v), Cripto, TACSTD2 (TROP2 or EGP1), CEA, Folate receptor 1, Mucin 16, Endothelin Receptor ETB, STEAP1, SLC44A4 (AGS-5), Nectin 4, AGS-16, Guanylyl cyclase C, Mucin 1, EGFRvIII, Mesothelin, IL2R, A33, Can, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGFR-1, VEGFR-2, VEGFR-3, TGFbeta, TGFbetaR, FGF, FGF8b, FGFR, PDGF, PDGFR, PDGFRalpha, PDGFRbeta, Ang-1, Ang-2, integrin, □2□1, alpha-v-beta3, alpha-v-beta5, alpha3beta1, alpha6beta4, alpha2beta1, anti-integrin alpha4, RANK-L, BLyS, c-MET, DR, DR10, TCRalpha, beta, ICOS, CTLA-4, CAIX (MN), EphA2, CA6, ovarian CA6, cervical CA6, breast CA6, angiopoietin-2, Cripto, ENPP3, Mesothelin, FOLR1, Nectin-4, TIM-1, Muc-16, Tissue Factor, LIV-1, GM2, alpha5 integrin, TLR-7, PD-1, AFP, CA125 (MUC16), Sialyl Lewis^(Y), CAMPATH-1, HLA-DR, anti-idiotype, carcinoembryonic antigen (CEA), TAG-72, Folate-binding protein, A33, G250, gangliosides (including GD2, GD3, GM2), Le^(Y), collagen 4 (collagen IV), collagen 18 (collagen XVIII), SC6, CA-125, CA19-9, p185^(HER2), de2-7 EGFR, Fibroblast activation protein (FAP), Tenascin, metalloproteinases, Endosialin, Carbonic anhydrase, Galectin 9, Aldolase A, elFgamma4, Tyrosinase, Galectin 4, HERKV-K10, p53, NY-LU-12, Restin, NY-CO-38, MAGE-1, MAGE-4a, SSX2, NY-ESO-1, SCP-1, HGFR, PTK 7, CCK-4, PDGFR, PTP-LAR, CDCP1, CADM1, IGSF4, Lu, BCAM, CEACAM6, JAM-A, PTGFRN (CD9P-1), MCAM, MUC18, MCP, EMMPRIN, TfR, TRAILR2, C1qR, hTERT, Survivin, MDM2, CYP1B1, Melan-A, MART-1, MART-2, Melanosomal proteins, gp100, neo-PAP, CDC27, MAGEs, WT1, MUM-1, MUM-2, MUM-3, BRAF, TPI, fibronectin, K-ras, beta-catenin, CDK4, caspase-8, p14^(ARF), p16^(INK4a), TGF□RII, bcr-abl, SYT-SSX, TRP-1, TRP-2, GnT-V, tyrosinase, FGF5, TEL-AML1, proteinase 3, HER2/neu, AFP, MUC-1, EBV-EBNA, HTLV-1 tax, HPV16-E7, mutated HLA-A2, HA1, SART3, GnT-V, CEACAM5, AGS-16, GPNMB, ESAT-6, RANK, CanAg, fibrin, TF, PRAME, CA19-9, CA50, CA125, CA195, CAM17.1/WGA, AFP, beta-MG, DU-PAN2, HE4, b-2 microglobulin, transferrin, transthyretin, ApoA1, TROP-2, CTLA-4, GITR, PD-1, PD-L1, c-KIT, CD11b-CD18 integrin heterodimer, DNA/Histon H1, Folate, EpCAM, Tenascin-c, ECM (proteoglycan or fibronectin), fibrinogen, SV40 large T antigen, SC6-Ag, SC-Ag, DR4 (death receptor 4), DR5, ESA, mucin, hPAM4, hRS7, HLA-DR, or CCR4, the antigen of general formula MTX1, MTX2, PECAN, thrombomodulin, Tn, cathepsin D, TYRO-3, MER or the antibody or antibody fragment is directed against a PF4/heparin complex.
 41. Method for preparing the products of formulae I, as defined in claim 26, wherein, if desired, a product of formula IA3

as well as the derivatives of the carboxylic acid function or functions such as the salts, esters or amides, preferably the amide formed with butanimide, where in formula IA3, X, A, X1, X2, X3, r, s, n₁, n₂ and n₃ have the meanings stated in claim 26, are reacted with a product of formula: H2N-L, or HO-L, in which L has the meaning stated in claim 26, in order to obtain a product of formula IA1:

product of formula IA1 which if desired is reacted with a product of formula: H2N-M, or HO-M, in which M has the meaning stated in claim 26, in order to obtain a product of formula IA2:

or product of formula IA3 which if desired is reacted with a residue of an -FL fluorescent radical in order to obtain a product of formula (IA4)

or product of formula IA3 which if desired is reacted with a residue of a radioactive radical —R* in order to obtain a product of formula (IA5)

products of formulae IA1 to IA5 corresponding to the products of formula IA which if desired are reacted with a product of formula P the definition of which is given in claim 26 in order to obtain a product of formula (IB):

in which W has the definition given in claim
 26. 42. Method of preparation according to claim 41, in which said products of formula IA1 to IA5 are attached to the protein P on at least one of its disulphide bridges by a reaction of nucleophilic substitution preferably carried out in a phosphate buffer, in the presence of TCEP.
 43. Method for treating a tumour, in particular colorectal cancer, hepatocarcinoma, a lung cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, colorectal cancer, K-RAS mutated colorectal cancer, a breast cancer, triple-negative breast cancer, metastatic breast cancer, liver cancer, head and neck cancer, Castleman disease, thyroid cancer, medulloblastoma, glioblastoma multiforme, glioma, sarcoma, anaplastic astrocytoma, kidney cancer, stomach cancer, malignant ascites, prostate cancer (metastatic or not), solid tumours, acute myeloid leukaemia, chronic lymphocytic leukaemia, melanoma, myeloma, multiple myeloma, lymphomas, mantle cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, comprising a step of administering a product of formula IB and in particular a product of formula IB2 according to claim
 35. 44. Method for treating an inflammatory or autoimmune disease, in particular graft rejection, rheumatoid arthritis, Crohn's disease, psoriasis, multiple sclerosis, cryopyrin-associated periodic syndromes, thrombocytopenic purpura, comprising a step of administering a product of formula IB and in particular a product of formula IB2 according to claim
 35. 45. Method for diagnostics or analysis involving detection of fluorescence by using products of formula I as defined in claim 26 constituted by the formulae IA:

and IB:

in which X, A, X1, X2, X3, n₁, n₂, n₃, r, s, t and W have the meanings stated in claim
 26. 46. Method for diagnostics or analysis involving detection of fluorescence by using products of formula IA4:

or of formula IB4

in which X, A, X1, X2, X3, n₁, n₂, n₃, r, s, t and FL have the meanings stated in claim 26 corresponding to the products of formula IA and IB as defined in claim 26 in which W represents an -FL fluorophore radical as defined in claim
 26. 47. Method for diagnostics or analysis involving detection of radioactivity by using products of formula (IA5):

or of formula (IB5):

in which X, A, X1, X2, X3, n₁, n₂, n₃, r, s, t and R* have the meanings stated in claim 26 corresponding to the products of formula IA and IB as defined in claim 26 in which W represents an R* radioactive radical.
 48. Method of diagnostics using products of formula (IA5) and (IB5) as defined in claim 47, in non-invasive imaging techniques, for measuring distribution and function in a biological system, of the PET (positron emission tomography) or SPECT (single photon emission computed tomography) type.
 49. Method for treating cancer comprising using products of formula (IA5) and (IB5) as defined in claim 27 as radiopharmaceuticals.
 50. Method for preparing the products of formula IB as defined in claim 26, wherein a product of formula IA

in which X is Br or I, A represents either an aryl or cycloalkyl radical, said aryl or cycloalkyl radicals being carbocyclic or heterocyclic, or A represents a

 group, a

 group or a

 group, in particular the

group is selected from the following radicals:

and X1, X2, X3, r, s, n₁, n₂, n₃, n₄ and W have the meanings stated in claim 26, is reacted with a product of formula P the definition of which is given in claim 26, in order to obtain a product of formula (IB):

in which t has the definition given in claim
 26. 